Lipidomic biomarkers for the prediction of cardiovascular outcomes in coronary artery disease patients not undergoing statin treatment

ABSTRACT

The present invention inter alia provides a method, and use thereof, of predicting severe CVD complications such as AMI or CVD death by detecting the lipid concentrations or lipid ratios of a biological sample and comparing it to a control and has identified specific lipid markers that are more specific and sensitive in predicting these CVD complications than currently utilized clinical markers. Also provided are antibodies towards said lipids, and the use thereof for predicting, diagnosing, preventing and/or treating CVD complications. The invention additionally relates to kits comprising lipids and/or an antibody thereto, for use in the prediction and/or diagnosis of CVD complications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of Ser. No. 14/356,594 filed on 6 May2014, which is the U.S. National Stage application of PCT/EP2012/071972filed 7 Nov. 2012, which claims priority to European patent application11188328.6 filed 8 Nov. 2011 and U.S. Provisional Patent Application61/556,909 filed 8 Nov. 2011, the entire disclosures of which are herebyincorporated herein by reference in their entireties.

DESCRIPTION Field of the Invention

This invention relates to methods and uses involving lipid levels topredict and prevent severe cardiovascular disease-associated fatalcomplications. The invention thus provides a means to identify and treathigh-risk coronary artery disease patients. The methods includeanalyzing lipid levels of a biological sample, and comparing it to acontrol.

BACKGROUND OF THE INVENTION

Worldwide, cardiovascular diseases (CVD) are among the leading causes ofmortality and morbidity with ever-increasing prevalence. CVD is used toclassify numerous conditions that affect the heart, heart valves, blood,and vasculature of the body. One of these conditions is coronary arterydisease (CAD). Statins are a family of cholesterol lowering drugs forpeople at high risk of cardiovascular complications. Statins are widelyused, as alone in the USA there are almost 20 million statin treatedpatients and it has been calculated that some 50 million patients wouldbenefit of statin treatment in the USA. However, despite statintreatment the CVD patients have risk to develop severe CVDcomplications. Early targeted initiation of preventive measures ofCVD-related fatal complications, such as acute myocardial infarction(AMI) and death, would be of great benefit and can provide a majoropportunity in reducing mortality and morbidity in patients sufferingfrom CVD. To this end, accurate identification of individuals who are atrisk of developing CVD complications is essential. However, traditionalrisk assessment fails to recognize a substantial proportion of patientsat high risk while a large proportion of individuals are classified ashaving intermediate risk, leaving patient management uncertain.Additional strategies to further refine risk assessment of high-risk CVDare therefore highly needed. To this end, the inventors have evaluatedthe role of novel lipidomic biomarkers as a prognostic tool for fatalcardiovascular events in CVD patients.

Statins are widely used drugs to prevent atherosclerotic end points inCVD patients and, therefore, a significant portion of middle agedpopulation is being treated with statins. Statins do lower efficientlyLDL-cholesterol and also many other lipids in the circulation. Thus,statin treatment is significantly affecting plasma concentrations ofmany potential lipidomic markers and therefore it is important toseparately study lipidomic biomarkers in subjects on statin treatmentand without statin treatment. It is known in the clinical practice thatconventional lipid biomarkers such as LDL-cholesterol are notinformative in statin treated patients, but these patients may stillhave a substantial residual risk of CAD complications despite statintreatment. This current invention deals with subjects who are notundergoing statin treatment at the time of the risk evaluation. A novelinnovative aspect here is that the investigators are studying riskmarkers separately in patients with type 2 Diabetes (DM2). DM2 iscausing numerous metabolic alterations in the human body and, therefore,DM2 may affect plasma levels of lipidomic biomarkers as well.Furthermore, CVD risk lipids in non-DM2 patients and DM2 patients maynot be the same and the prognostic accuracy can potentially be greatlyimproved if these subject groups are studied separately.

Plasma or serum total cholesterol, LDL-cholesterol or HDL-cholesterolconcentrations have been used as gold standard biomarkers for CVD/CADrisk prediction. However, a number of coronary artery disease (CAD) oracute myocardial infarction (AMI) patients have LDL-C levels within therecommended range suggesting the need for additional diagnostic measuresof the residual risk. It is evident from earlier large scale populationstudies that these measurements associate with the CAD risk and CADendpoints such as AMI or cardiovascular death. Therefore, preventivetreatment strategies have so far been addressed to lower LDL-Cconcentrations (mainly by statin treatment) and more recently alsoattempts to raise HDL-C have been made (e.g., by CETP-inhibitors). Onthe other hand, it has also been observed that one half of the AMIpatients actually do have normal LDL cholesterol levels and that thereis a substantial residual risk in statin treated patients despite aLDL-C lowering. Furthermore, recent publications have demonstrated thatplasma levels of apolipoprotein B (apoB), the main surface protein onLDL particles, and LDL-C, the amount of cholesterol in those particles,are correlated and, considered separately, as positive risk factors.Plasma levels of apolipoprotein A₁, the main surface protein on HDLparticles, and HDL-C, the amount of cholesterol in those particles, arealso correlated with each other and, considered separately, as negativerisk factors. Importantly, for a given usual apoB, lower LDL-C has beenobserved to associate with a higher risk of AMI supporting the viewthat, on average, LDL particles with low cholesterol content perparticle (small, dense LDL particles) are particularly hazardous. Thus,it seems possible that LDL-C associates directly with the more dangerousmolecules carried by LDL-particles and that LDL-C is only an indirectmeasurement of the risk. Therefore, it is of importance to search formolecules e.g., certain lipid species that are directly related withhazardous (i.e., fatal) cardiovascular events.

Lipid metabolite imbalance is a probable cause of dyslipidemia and theensuing atherosclerosis manifested in its gravest form as the vulnerableatherosclerotic plaque. Atherosclerotic plaques are complex molecularformations that contain numerous lipids. However, there are otherfactors than lipid rich plaques or LDL cholesterol that make lipids anattractive group of molecules for CVD studies. Lipids are tightlyregulated which makes Lipidomic data robust and informative on thecurrent state of the studied organism. Also, lipids are one of theculmination points of a biological system, more the true outcome thanthe predictor. Combining Lipidomic data with appropriate biobankedclinical material presents a good opportunity for biomarker discovery.Moreover, lipidomics can be used as a gauge of efficacy and safety indrug development and evolving theragnostics. Lipidomic biomarkers areprime candidates for true companion diagnostics in the CVD area andpresent many opportunities for improved translational medicine as well.

The plaque building blocks and lipoprotein components that are thoughtto traffic lipids to the site of lesion formation can now be resolvedwith Lipidomic studies correlating lipid structure and composition tofunction and thereby disease pathogenesis. The number of lipid mediatorsin the human body is overwhelming. Their identification andquantification is facilitated by the advances in mass spectrometry andlipid biochemistry, which enable the simultaneous high throughputidentification and quantification of hundreds of molecular lipid speciesin several lipid classes (Ejsing C S, et al: Global analysis of theyeast lipidome by quantitative shotgun mass spectrometry. Proc Natl AcadSci USA 2009, 106:2136-2141; Stahlman M, et al: High-throughput shotgunlipidomics by quadrupole time-of-flight mass spectrometry. J ChromatogrB Analyt Technol Biomed Life Sci 2009 Hiukka A, et al: ApoCIII-enrichedLDL in type 2 diabetes displays altered lipid composition, increasedsusceptibility for sphingomyelinase, and increased binding to biglycan.Diabetes 2009, 58:2018-2026; Linden D, et al: Liver-directedoverexpression of mitochondrial glycerol-3-phosphate acyltransferaseresults in hepatic steatosis, increased triacylglycerol secretion andreduced fatty acid oxidation. FASEB J 2006, 20:434-443.) collectivelyreferred to as the lipidome. Lipidomic studies identify lipid cellulardistribution and describe their biochemical mechanisms, interactions anddynamics. Importantly, lipidomics quantifies the exact chemicalcomposition of lipidomes (Han X, Gross R W: Global analyses of cellularlipidomes directly from crude extracts of biological samples by ESI massspectrometry: a bridge to lipidomics. J Lipid Res 2003, 44:1071-1079).

Due to both high sensitivity and selectivity of lipidomics, even thesmallest sample amounts can be analyzed today. The bulk of the lipiddata in the art today presents lipids in a sum composition format, i.e.phosphatidylcholine (PC) 34:1 (Brugger B, et al: Quantitative analysisof biological membrane lipids at the low picomole level bynano-electrospray ionization tandem mass spectrometry. Proc Natl AcadSci USA 1997, 94:2339-2344) where the molecular lipid and the attachedfatty acid tails remain unidentified. The identification of molecularlipid species, e.g., PC 16:0/18:1 (Ekroos K, et al: Charting molecularcomposition of phosphatidylcholines by fatty acid scanning and ion trapMS3 fragmentation. J Lipid Res 2003, 44:2181-2192) is the main featureof advanced lipidomics, which delivers highly resolved molecular lipidspecies rather than summed fatty acid information. For example, theinformation of the type of fatty acids and their positions of attachmentto the glycerol backbone making up the particular PC molecule isrevealed. There are conventional techniques such as thin-layerchromatography combined with gas chromatography but they not onlyrequire considerably larger sample amounts and laborious samplepreparation, but they do not deliver the molecular lipid species.Despite multiple mass spectrometry techniques capable of characterizinglipid entities, most of them are still unable to deliver reliablehigh-quality quantitative data in terms of absolute or close-to absoluteconcentrations. In the context of the present invention, electrosprayionization mass spectrometry-based lipidomics is the preferredtechnology and can utilize both shotgun and targeted lipidomics forexhaustive deciphering and precise quantification of molecularlipidomes. The superior quality and specificity of shotgun and targetedlipidomics will meet stringent regulatory standards, such as goodlaboratory practice guidelines (GLP) when set-up in the properenvironment. Using these technologies quantification of up to twothousand molecular lipids is possible even in a high throughput format.

Lipidomics is a tool for differentiating patients based on theirmolecular lipid profiles. Personalized medicine and diagnostics enabledby lipidomics will facilitate the mission of the right individualreceiving the right drug at the right time and dose. Several worksemploying analytes consisting of lipids, proteins and hydrophilicmolecules among many others have been conducted to meet the needs ofpersonalized medicine. Recently, non-hypothesis-driven metabolomicscreenings have been used to identify novel CVD biomarkers.

For example, WO2004/038381 discloses a method for metabolomicallyfacilitating the diagnosis of a disease state of a subject, or forpredicting whether a subject is predisposed to having a disease statewherein the small molecule profile from a subject is obtained andcompared to a standard small molecule profile.

WO2008/148857 discloses a method to assess the risk of cardiovasculardisease in a patient (including atherosclerosis) by isolating the HDLfraction and sub-fraction from a blood sample of the patient. Thecomponents of the HDL fraction or sub-fraction to be measured wereSphingosine-1-Phosphate (S1P), sphingomyelin (SM) and Apolipoprotein A-I(apoA-1).

WO2008/11943 further discloses markers for detecting coronary arterydisease that can indicate a patient at risk of having or developingcoronary artery disease. These include 15 “first-choice” molecules whichwere: C18:3 Cholesterol ester, C32:1 Phosphatidylcholine, Alanine, Lipid(mainly VLDL), Lysine, Hexadecanoic acid, C36:2 Phosphatidylcholine,Formate, C32:2 Phosphatidylcholine, C18:2 (Linoleic Acid), Cholesterol,C18:2 Lyso-phosphatidylcholine, C36:3 Phosphatidylcholine, C34:4Phosphatidylcholine and C34:3 Phosphatidylcholine.

Furthermore, US2007/0099242 describes a method to determine if a subjectis at risk to develop, or is suffering from cardiovascular disease. Themethod involves determining a change in the amount of a biomarker in thebiological sample or HDL sub-fraction thereof, compared to a controlsample, wherein the biomarker is at least one of Apolipoprotein C-IV(“ApoC-IV”), Paraoxonase 1 (“PON-1”), Complement Factor 3 (“C3”),Apolipoprotein A-IV (“ApoA-IV”), Apolipoprotein E (“ApoE”),Apolipoprotein LI (“ApoL 1”), Complement Factor C4 (“C4”), ComplementFactor C4B1 (“C4B1”), Histone H2A, Apolipoprotein C-II (“ApoC-II”),Apolipoprotein M (“ApoM”), Vitronectin, Haptoglobin-related Protein andClusterin. The document also discloses a method for detecting thepresence of one or more atherosclerotic lesions wherein a change in theamount of a biomarker in the biological sample or HDL sub-fractionthereof is detected, compared to a control sample and wherein thebiomarker is selected from PON-1, C3, C4, ApoE, ApoM and C4B1. Allbiomarkers mentioned in this document are protein or lipoproteinbiomarkers.

WO2011/063470 compares the lipid profiles of patients with coronarydisease (stable) with patients with acute coronary syndrome (ACS) havingacute chest pain, ECG changes and troponin I elevations. This comparisonrevealed lipid markers that associate with troponin I and clinicalmarkers of ACS suggesting that lipids may be used as a biomarker ofacute myocardial ischemia. However, in acute cardiovascular setting,troponin I seems to be superior marker compared to lipid profiles(Meikle et al. Plasma lipidomic analysis of stable and unstable coronaryartery disease. Arterioscler Thromb Vasc Biol. 2011 November;31(11):2723-32.) and the findings do not predict patient outcome norlong-term risk of acute myocardial ischemia or cardiovascular death.

From previous work it cannot be extrapolated that lipid analysis willyield by default a CVD biomarker predictive to the fatal outcomesassociated with CVD/CAD. There remains a need for specific markersuseful for identifying specific risk patient populations within patientsgenerally suffering from or being at risk of CVD/CAD.

The present invention identifies biomarkers of high risk CVD byabsolute, or close to absolute, quantification of defined molecularlipid species instead of profiling multiple analytes. Importantly, whilemany of the existing biomarker candidates are composite fingerprints ofmultiple factors, the lipidomics approach herein shows value already ata level of single species or ratios thereof. The present applicationdiscloses an improved lipid assay approach over those in the prior artsince it takes into account factors that affect lipid metabolism such aslipid lowering treatment (e.g. statins) and diabetes. Therefore, thepresent application provides novel personalized prediction markers.

SUMMARY OF THE INVENTION

The present invention provides novel lipidomic markers for predictingand preventing severe CVD/CAD-associated complications, including AMI,stroke and death, in CVD/CAD patients not undergoing statin treatment.These markers thus provide a means to identify and treat high-riskcoronary artery disease patients. Specifically, it has been found thatthe lipid molecules, lipid-lipid concentration ratios and lipid-clinicalconcentration ratios provided herein, when displaying an increased ordecreased level—as the case may be—in samples from CAD patients, areuseful lipidomic markers for the methods and uses in accordance with thepresent invention. These sensitive and specific markers werespecifically tested to display superior diagnostic and prognostic valuecompared to the current clinically-used markers predictive for CVD/CADoutcomes. In fact, the currently available biomarkers such as LDL-C orHDL-C have only very limited or no value in predicting the CVD deathrisk in CAD patients. The present invention therefore represents asignificant advantage to other markers which are currently used todiagnose and/or predict CVD and CVD complications, which include LDL-C,total plasma/serum cholesterol and Apolipoprotein B and A1. Thus, thelipidomic markers provided herein allow better diagnosis of orassessment of the risk to develop major CVD complications such as AMI orCVD death.

In accordance with the present invention, methods are inter aliadisclosed herein for determining the risk of a CVD patient notundergoing statin treatment to develop CVD complications, or fordetermining warning signs of CVD risks, (including death, myocardialinfarction (MI), angina pectoris, transischemic attack (TIA) and stroke)in said patient.

Methods according to the invention typically comprise the steps of: a)providing a biological sample from a CAD subject; b) determining a lipidconcentration, lipid-lipid concentration ratio, or lipid-clinicalconcentration ratio or (a) corresponding profile(s) from said sample(i.e., determining information on a lipidomic marker in accordance withthe invention); and c) comparing said determined lipid concentration,lipid-lipid concentration ratio, or lipid-clinical concentration ratioor said corresponding profile(s) to the corresponding lipidconcentration, lipid-lipid concentration ratio, or lipid-clinicalconcentration ratio or the corresponding profile(s) in a control.

As mentioned above, the lipidomic marker to be compared between thesubject sample and the control (or control sample) may be one or more ofthe lipid concentration(s), lipid-lipid concentration ratio(s), orlipid-clinical concentration ratio(s) or combinations thereof, i.e., thecorresponding profile(s), as described and claimed herein. In thisregard, the control or control sample allows establishment of thelipidomic marker baseline or starting point.

The lipidomic markers of the present invention allow for prediction andprevention of fatal CVD complications. This will facilitate earlierintervention, less symptom development and suffering and decreasedmorbidity/mortality associated with CVD. Thus, the lipidomic markersdescribed and claimed herein allow for individual tailoring of drugintervention for patients being at risk to develop major CVDcomplications.

In other words, the present invention discloses diagnostic and/orpredictive lipid markers and lipid-lipid or lipid-clinical concentrationratios for use in predicting CVD complications such as AMI or CVD deathin CVD patients who are not undergoing statin treatment. The inventionuses the measurement of lipid concentrations, lipid-lipid and/orlipid-clinical concentration ratios to determine the risk of saidsubject to develop CVD complications such as AMI and/or CVD death. Thesubject may have previously suffered from a cardiovascular disease eventsuch as angina pectoris, myocardial infarction or stroke.

Accordingly, in one aspect of the invention, a method is provided fordetermining whether a subject who is not undergoing statin treatment isat risk to develop one or more CVD complications such as AMI and/or CVDdeath, said method comprising determining in a sample from said subjectone or more lipid-lipid concentration ratio(s), wherein (an) increasedor decreased lipid-lipid concentration ratio(s) in said sample, whencompared to a control sample, is (are) indicative of said subject havingan increased risk of developing one or more CVD complications, such asAMI or CVD death, wherein the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Cer(d18:1/18:0)/Cer(d18:1/24:0),Cer(d18:1/20:0)/Cer(d18:1/24:0), Cer(d18:1/22:0)/Cer(d18:1/24:0)Gb3(d18:1/16:0)/PC 18:0/22:6, SM (d18:1/16:0) (d18:1/15:1-OH)/SM(d18:1/24:0) (d18:1/23:1-OH) (Table 3);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/PC 16:0/18:2, SM (d18:1/24:0) (d18:1/23:1-OH)/SM(d18:1/24:1) (d18:1/23:2-OH), Cer(d18:1/24:0)/Cer(d18:1/24:1),Cer(d18:1/24:0)/SM (d18:1/14:0) (d18:1/13:1-OH),Cer(d18:1/24:0)/Gb3(d18:1/16:0) and Cer(d18:1/24:0)/SM (d18:1/16:1)(d18:1/15:2-OH) (Table 3).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: Cer(d18:1/24:0)/PC 16:0/18:2, SM (d18:1/24:0)(d18:1/23:1-OH)/SM (d18:1/24:1) (d18:1/23:2-OH), Cer(d18:1/24:0)/SM(d18:1/14:0) (d18:1/13:1-OH), Cer(d18:1/24:0)/Gb3(d18:1/16:0) andCer(d18:1/24:0)/SM (d18:1/16:1) (d18:1/15:2-OH) (Table 3).

In yet another alternative embodiment, the present invention relates toa method for determining whether a subject who is not undergoing statintreatment is at risk to develop one or more CVD complications, such asAMI and/or CVD death, comprising determining in a sample from saidsubject one or more lipid-clinical concentration ratio(s), wherein (an)increased or decreased lipid-clinical concentration ratio(s) in saidsample, when compared to a control sample, is (are) indicative of saidsubject having an increased risk of developing one or more CVDcomplications, such as AMI or CVD death, wherein the one or morelipid-clinical concentration ratio(s) whose increase(s) is (are)compared to the control is (are) selected from: GlcCer(d18:1/16:0)/totalcholesterol, Gb3(d18:1/16:0)/apolipoprotein A-I,Gb3(d18:1/18:0)/apolipoprotein A-I, Gb3(d18:1/16:0)/HDL cholesterol andPC 16:0/18:2/total cholesterol (Table 3);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/supersensitive C-reactive protein and PC18:0/22:6/supersensitive C-reactive protein (Table 3).

For the purposes of the invention, and particularly for lipid-clinicalconcentration ratios, an Apolipoprotein A-I measurement mayalternatively be an Apolipoprotein A-II measurement.

In another aspect of the invention, a method is provided for determiningwhether a subject not undergoing statin treatment who is not sufferingfrom type 2 diabetes mellitus is at risk to develop one or more CVDcomplications, such as acute myocardial infarction (AMI) and/or CVDdeath, said method comprising determining in a sample from said subjectthe concentration(s) of one or more lipid(s), wherein (an) increased ordecreased concentration(s) in said sample, when compared to a controlsample, is (are) indicative of said subject having an increased risk ofdeveloping one or more CVD complications, such as AMI or CVD death,wherein the one or more lipid(s) whose increase(s) in concentration is(are) compared to the control is (are) selected from: CE 19:1 oxCE682.6, GlcCer(d18:1/16:0), SM (d18:1/18:1), CE 20:4, LacCer(d18:1/16:0),Cer 18:1/16:0, SM 18:1/16:0 and CE 16:0 (Tables 4a, 6 and 8);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PC 18:0/22:6,SM (d18:1/23:1) (d18:1/22:2-OH), PC 16:0/22:6, SM 18:1/24:0 and LPC 16:0(Tables 4a, 6 and 8).

In one particular embodiment, the one or more lipid(s) whose increase(s)in concentration is (are) compared to the control is (are) selectedfrom: CE 19:1 (oxCE 682.6), CE 20:4, Cer 18:1/16:0 and SM 18:1/16:0(Tables 4a, 6 and 8);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PC 18:0/22:6,SM 18:1/24:0 and LPC 16:0 (Tables 4a, 6 and 8).

In a preferred embodiment, the one or more lipid(s) whose increase(s) inconcentration is (are) compared to the control is (are) selected from:GlcCer(d18:1/16:0), CE 20:4, LacCer(d18:1/16:0), Cer 18:1/16:0, SM18:1/16:0 and CE 16:0 (Table 8);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: PC 16:0/22:6, SM18:1/24:0 and LPC 16:0 (Table 8).

In one particularly preferred embodiment, the one or more lipid(s) whoseincrease(s) in concentration is (are) compared to the control is (are)selected from: CE 20:4, Cer 18:1/16:0 and SM 18:1/16:0 (Table 8); andthe one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: SM 18:1/24:0 and LPC16:0 (Table 8).

In an alternative embodiment, the present invention relates to a methodfor determining whether a subject not undergoing statin treatment who isnot suffering from type 2 diabetes mellitus is at risk to develop one ormore CVD complications such as AMI and/or CVD death, comprisingdetermining in a sample from said subject one or more lipid-lipidconcentration ratio(s), wherein (an) increased or decreased lipid-lipidconcentration ratio(s) in said sample, when compared to a controlsample, is (are) indicative of said subject having an increased risk ofdeveloping one or more CVD complications, such as AMI or CVD death,wherein the one or more lipid-lipid concentration ratio(s) whoseincrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/22:2-OH), Cer(d18:1/16:0)/PC18:0/22:6, Cer(d18:1/18:0)/PC 16:0/22:6, GlcCer(d18:1/16:0)/PC18:0/22:6, GlcCer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/22:2-OH),Cer(d18:1/16:0)/PC 16:0/22:6, Cer(d18:1/20:0)/SM (d18:1/23:1)(d18:1/22:2-OH), GlcCer(d18:1/18:0)/PC 16:0/22:6, SM (d18:1/18:1)/SM(d18:1/23:1) (d18:1/22:2-OH), Cer(d18:1/20:0)/PC 16:0/22:6, SM(d18:1/16:0) (d18:1/15:1-OH)/SM (d18:1/23:1) (d18:1/22:2-OH), SM(d18:1/15:0) (d18:1/14:1-OH)/SM (d18:1/23:1) (d18:1/22:2-OH),GlcCer(d18:1/20:0)/SM (d18:1/23:1) (d18:1/22:2-OH), Cer(d18:1/24:1)/PC18:0/22:6, SM (d18:1/16:1) (d18:1/15:2-OH)/SM (d18:1/23:1)(d18:1/22:2-OH), GlcCer(d18:1/16:0)/PC 16:0/22:6, LacCer(d18:1/22:0)/PC16:0/22:6, LacCer(d18:1/22:0)/SM (d18:1/23:1) (d18:1/22:2-OH),GlcCer(d18:1/20:0)/PC 16:0/22:6, CE 16:0/PC 18:0/22:6,Cer(d18:1/22:0)/PC 18:0/22:6, CE 18:2/PC 18:0/22:6, CE 18:1/PC18:0/22:6, Cer(d18:1/16:0)/Cer(d18:1/24:0), Gb3(d18:1/16:0)/PC16:0/22:6, CE 20:4/PC 18:0/22:6, CE 22:6/PC 18:0/22:6, PC 16:0/16:0/PC16:0/22:6, CE 16:0/PC 16:0/22:6, CE 18:2/PC 16:0/22:6,Gb3(d18:1/24:0)/PC 16:0/22:6, CE 18:1/PC 16:0/22:6, CE 20:4/PC16:0/22:6, SM (d18:1/16:1) (d18:1/15:2-OH)/SM (d18:1/24:0)(d18:1/23:1-OH), Cer(d18:1/16:0)/Cer(d18:1/22:0), CE16:0/Cer(d18:1/24:0), SM (d18:1/18:1)/SM (d18:1/24:0) (d18:1/23:1-OH),GlcCer(d18:1/18:0)/SM (d18:1/24:0) (d18:1/23:1-OH) and SM (d18:1/15:0)(d18:1/14:1-OH)/SM (d18:1/24:0) (d18:1/23:1-OH)(Table 4b);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/GlcCer(d18:1/18:0), Cer(d18:1/24:0)/GlcCer(d18:1/16:0),PC 16:0/22:6/SM (d18:1/16:0) (d18:1/15:1-OH), PC 18:0/22:6/SM(d18:1/14:0) (d18:1/13:1-OH), PC 16:0/22:6/SM (d18:1/16:1)(d18:1/15:2-OH), PC 18:0/22:6/SM (d18:1/16:0) (d18:1/15:1-OH), PC18:0/22:6/SM (d18:1/15:0) (d18:1/14:1-OH), PC 18:0/22:6/SM (d18:1/18:0),PC 18:0/22:6/SM (d18:1/16:1) (d18:1/15:2-OH) and PC 18:0/22:6/SM(d18:1/18:1) (Table 4b).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Cer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/22:2-OH),Cer(d18:1/16:0)/PC 18:0/22:6, Cer(d18:1/18:0)/PC 16:0/22:6,GlcCer(d18:1/16:0)/PC 18:0/22:6, GlcCer(d18:1/18:0)/SM (d18:1/23:1)(d18:1/22:2-OH), Cer(d18:1/16:0)/PC 16:0/22:6, Cer(d18:1/20:0)/SM(d18:1/23:1) (d18:1/22:2-OH), GlcCer(d18:1/18:0)/PC 16:0/22:6, SM(d18:1/18:1)/SM (d18:1/23:1) (d18:1/22:2-OH), Cer(d18:1/20:0)/PC16:0/22:6, SM (d18:1/16:0) (d18:1/15:1-OH)/SM (d18:1/23:1)(d18:1/22:2-OH), SM (d18:1/15:0) (d18:1/14:1-OH)/SM (d18:1/23:1)(d18:1/22:2-OH), GlcCer(d18:1/20:0)/SM (d18:1/23:1) (d18:1/22:2-OH),Cer(d18:1/24:1)/PC 18:0/22:6, SM (d18:1/16:1) (d18:1/15:2-OH)/SM(d18:1/23:1) (d18:1/22:2-OH), GlcCer(d18:1/16:0)/PC 16:0/22:6,LacCer(d18:1/22:0)/PC 16:0/22:6, CE 16:0/PC 18:0/22:6, CE 18:2/PC18:0/22:6 and Cer(d18:1/16:0)/Cer(d18:1/24:0) (Table 6);

and the one or more lipid-lipid concentration ratio(s) whose decrease(s)is (are) compared to the control is (are) selected from: PC 18:0/22:6/SM(d18:1/16:1) (d18:1/15:2-OH) and PC 18:0/22:6/SM (d18:1/18:1)(Table 6).

In yet another alternative embodiment the present invention relates to amethod for determining whether a subject not undergoing statin treatmentwho is not suffering from type 2 diabetes mellitus is at risk to developone or more CVD complications, such as AMI and/or CVD death, comprisingdetermining in a sample from said subject one or more lipid-clinicalconcentration ratio(s), wherein (an) increased or decreasedlipid-clinical concentration ratio(s) in said sample, when compared to acontrol sample, is (are) indicative of said subject having an increasedrisk of developing one or more CVD complications, such as AMI or CVDdeath, wherein the one or more lipid-clinical concentration ratio(s)whose increase(s) is (are) compared to the control is (are) selectedfrom: Cer(d18:1/16:0)/HDL cholesterol, GlcCer(d18:1/16:0)/apolipoproteinA-I, CE 19:1 oxCE 682.6/apolipoprotein A-I,GlcCer(d18:1/18:0)/apolipoprotein A-I, GlcCer(d18:1/18:0)/HDLcholesterol, GlcCer(d18:1/16:0)/HDL cholesterol,GlcCer(d18:1/20:0)/apolipoprotein A-I, GlcCer(d18:1/18:0)/totalcholesterol and Cer(d18:1/16:0)/apolipoprotein B

(Table 4c);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:LacCer(d18:1/24:0)/supersensitive C-reactive protein, CE17:1/supersensitive C-reactive protein, SM (d18:1/16:0)(d18:1/15:1-OH)/supersensitive C-reactive protein,GlcCer(d18:1/24:0)/supersensitive C-reactive protein,Gb3(d18:1/22:0)/supersensitive C-reactive protein, PC18:0/20:3/apolipoprotein B, PC 16:0/16:1/supersensitive C-reactiveprotein, PC 16:0/22:6/total cholesterol, PC 16:0/22:6/apolipoprotein B,PC 18:1/18:1/supersensitive C-reactive protein, SM 8:1/24:1)(d18:1/23:2-OH)/supersensitive C-reactive protein, PC18:0/18:2/supersensitive C-reactive protein,Cer(d18:1/24:1)/supersensitive C-reactive protein,Cer(d18:1/22:0)/supersensitive C-reactive protein, PC18:0/22:6/triglycerides, SM (d18:1/18:0)/supersensitive C-reactiveprotein, PC 18:0/20:3/supersensitive C-reactive protein, SM (d18:1/24:0)(d18:1/23:1-OH)/supersensitive C-reactive protein, PC16:0/18:1/supersensitive C-reactive protein, SM 8:1/23:1)(d18:1/22:2-OH)/total cholesterol, PC 18:0/22:6/LDL cholesterol, PC18:0/22:6/total cholesterol, SM (d18:1/23:1) (d18:1/22:2-OH)/LDLcholesterol, SM (d18:1/23:1) (d18:1/22:2-OH)/apolipoprotein B, PC18:0/22:6/apolipoprotein B, SM (d18:1/23:0)(d18:1/22:1-OH)/supersensitive C-reactive protein, PC18:0/18:1/supersensitive C-reactive protein, LPC 16:0/supersensitiveC-reactive protein, PC 16:0/22:6/supersensitive C-reactive protein, SM(d18:1/23:1) (d18:1/22:2-OH)/supersensitive C-reactive protein, PC18:0/22:6/lipoprotein(a) and PC 16:0/18:0/supersensitive C-reactiveprotein (Table 4c).

In one particular embodiment, the one or more lipid-clinicalconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is (are) selected from: CE 19:1 oxCE 682.6/apolipoprotein A-I,GlcCer(d18:1/18:0)/HDL cholesterol, GlcCer(d18:1/16:0)/HDL cholesterol,GlcCer(d18:1/20:0)/apolipoprotein A-I and GlcCer(d18:1/18:0)/totalcholesterol (Table 4c).

In a preferred embodiment, the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Cer(d18:1/16:0)/HDL cholesterol (Table 6);

and/or the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: PC18:0/22:6/LDL cholesterol, PC 18:0/22:6/total cholesterol and PC18:0/22:6/apolipoprotein B (Table 6).

In another aspect of the invention, a method is provided for determiningwhether a subject not undergoing statin treatment who is suffering fromtype 2 diabetes mellitus is at risk to develop one or more CVDcomplications, such as acute myocardial infarction (AMI) and/or CVDdeath, said method comprising determining in a sample from said subjectthe concentration(s) of one or more lipid(s), wherein (an) increased ordecreased concentration(s) in said sample, when compared to a controlsample, is (are) indicative of said subject having an increased risk ofdeveloping one or more CVD complications, such as AMI or CVD death,wherein the one or more lipid(s) whose increase(s) in concentration is(are) compared to the control is (are) selected from: Gb3(d18:1/24:1),Gb3(d18:1/16:0), GlcCer 18:1/16:0, LacCer 18:1/16:0 and PC 16:0/22:6(Tables 5a, 7 and 8);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PCO-16:0/20:4-alkyl, CE 20:4, CE 18:0, CE 14:0, CE 22:6, CE 18:3, GlcCer18:1/18:0 and SM 18:1/24:0 (Tables 5a, 7 and 8).

In one particular embodiment, the one or more lipid(s) whose increase(s)in concentration is (are) compared to the control is (are) selectedfrom: Gb3(d18:1/24:1), Gb3(d18:1/16:0), GlcCer 18:1/16:0 and LacCer18:1/16:0 (Tables 5a, 7 and 8);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PCO-16:0/20:4-alkyl, CE 20:4, CE 22:6, GlcCer 18:1/18:0 and SM 18:1/24:0(Tables 5a, 7 and 8).

In a preferred embodiment, the one or more lipid(s) whose increase(s) inconcentration is (are) compared to the control is (are) selected from:Gb3 18:1/16:0, GlcCer 18:1/16:0, LacCer 18:1/16:0 and PC 16:0/22:6(Table 8);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: CE 14:0, CE 22:6, CE18:3, GlcCer 18:1/18:0 and SM 18:1/24:0 (Table 8).

In one particularly preferred embodiment, the one or more lipid(s) whoseincrease(s) in concentration is (are) compared to the control is (are)selected from: Gb3 18:1/16:0, GlcCer 18:1/16:0 and LacCer 18:1/16:0(Table 8);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: CE 22:6, GlcCer18:1/18:0 and SM 18:1/24:0 (Table 8).

In an alternative embodiment, the present invention relates to a methodfor determining whether a subject not undergoing statin treatment who issuffering from type 2 diabetes mellitus is at risk to develop one ormore CVD complications such as AMI and/or CVD death, comprisingdetermining in a sample from said subject one or more lipid-lipidconcentration ratio(s), wherein (an) increased or decreased lipid-lipidconcentration ratio(s) in said sample, when compared to a controlsample, is (are) indicative of said subject having an increased risk ofdeveloping one or more CVD complications, such as AMI or CVD death,wherein the one or more lipid-lipid concentration ratio(s) whoseincrease(s) is (are) compared to the control is (are) selected from:Gb3(d18:1/24:1)/SM (d18:1/17:0) (d18:1/16:1-OH), Gb3(d18:1/24:1)/PCO-16:0/20:4-alkyl, Gb3(d18:1/16:0)/SM (d18:1/17:0) (d18:1/16:1-OH),Gb3(d18:1/24:1)/GlcCer(d18:1/24:0), Gb3(d18:1/24:1)/GlcCer(d18:1/22:0)and Gb3(d18:1/22:0)/SM (d18:1/17:0) (d18:1/16:1-OH)

(Table 5b);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: CE18:0/PC 18:0/18:2, PC 18:0/18:2/PE 18:0/18:2, Cer(d18:1/24:0)/PE18:0/18:2, CE 18:0/CE 18:1, CE 18:2/Gb3(d18:1/16:0), CE16:0/Gb3(d18:1/16:0), CE 18:0/PC 16:0/22:6, CE 14:0/Gb3(d18:1/24:0), CE18:0/SM (d18:1/16:0) (d18:1/15:1-OH), CE 18:3/SM (d18:1/14:0)(d18:1/13:1-OH), CE 18:0/PC 16:0/18:2, Cer(d18:1/24:0)/PCO-16:0/18:1-alkyl, CE 18:0/Gb3(d18:1/24:0), CE 18:3/PC 16:0/18:1, PC18:0/20:3/PC O-16:0/18:1-alkyl, CE 14:0/PC 16:0/16:0, CE17:1/Gb3(d18:1/16:0), CE 14:0/SM (d18:1/14:0) (d18:1/13:1-OH), PC18:0/20:3/PE 18:0/18:2, CE 18:0/SM (d18:1/14:0) (d18:1/13:1-OH), CE14:0/PC 16:0/18:1, CE 18:0/SM (d18:1/24:1) (d18:1/23:2-OH), CE18:0/Cer(d18:1/16:0), CE 18:0/Cer(d18:1/24:1), CE 18:0/PC 16:0/16:0, CE18:0/PC 18:1/18:1, CE 18:0/PC 16:0/18:1, CE 20:4/PC O-16:0/18:1-alkyl,CE 18:3/PE 18:0/18:2, CE 20:4/Gb3(d18:1/16:0), CE 14:0/PE 18:0/18:2, CE18:0/Cer(d18:1/26:1), CE 18:3/Gb3(d18:1/22:0), CE 14:0/Gb3(d18:1/16:0),CE 18:3/PC O-16:0/18:1-alkyl, CE 18:3/Gb3(d18:1/16:0), CE 14:0/PCO-16:0/18:1-alkyl, CE 20:4/Gb3(d18:1/18:0), CE 18:3/Gb3(d18:1/24:1), CE14:0/Gb3(d18:1/24:1), CE 20:5/PC O-16:0/18:1-alkyl, CE18:0/Gb3(d18:1/18:0), CE 18:0/Gb3(d18:1/16:0) and CE18:0/Gb3(d18:1/24:1) (Table 5b).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: CE 18:0/PC 18:0/18:2, PC 18:0/18:2/PE 18:0/18:2,Cer(d18:1/24:0)/PE 18:0/18:2, CE 18:0/CE 18:1, CE 18:2/Gb3(d18:1/16:0),CE 16:0/Gb3(d18:1/16:0), CE 18:0/PC 16:0/22:6, CE 14:0/Gb3(d18:1/24:0),CE 18:0/SM (d18:1/16:0) (d18:1/15:1-OH), CE 18:3/SM (d18:1/14:0)(d18:1/13:1-OH), CE 18:0/PC 16:0/18:2, Cer(d18:1/24:0)/PCO-16:0/18:1-alkyl, CE 18:0/Gb3(d18:1/24:0), CE 18:3/PC 16:0/18:1, PC18:0/20:3/PC O-16:0/18:1-alkyl, CE 14:0/PC 16:0/16:0, CE17:1/Gb3(d18:1/16:0), CE 14:0/SM (d18:1/14:0) (d18:1/13:1-OH), PC18:0/20:3/PE 18:0/18:2, CE 18:0/SM (d18:1/14:0) (d18:1/13:1-OH), CE14:0/PC 16:0/18:1, CE 18:0/SM (d18:1/24:1) (d18:1/23:2-OH), CE18:0/Cer(d18:1/24:1), CE 18:0/PC 16:0/16:0, CE 18:0/PC 18:1/18:1, CE18:0/PC 16:0/18:1, CE 20:4/PC O-16:0/18:1-alkyl, CE 18:3/PE 18:0/18:2,CE 20:4/Gb3(d18:1/16:0), CE 14:0/PE 18:0/18:2, CE 18:0/Cer(d18:1/26:1),CE 18:3/Gb3(d18:1/22:0), CE 14:0/Gb3(d18:1/16:0), CE 18:3/PC0-16:0/18:1-alkyl, CE 18:3/Gb3(d18:1/16:0), CE 14:0/PCO-16:0/18:1-alkyl, CE 20:4/Gb3(d18:1/18:0), CE 18:3/Gb3(d18:1/24:1), CE14:0/Gb3(d18:1/24:1), CE 20:5/PC O-16:0/18:1-alkyl, CE18:0/Gb3(d18:1/18:0), CE 18:0/Gb3(d18:1/16:0) and CE18:0/Gb3(d18:1/24:1) (Table 5b).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Gb3(d18:1/24:1)/SM (d18:1/17:0) (d18:1/16:1-OH),Gb3(d18:1/16:0)/SM (d18:1/17:0) (d18:1/16:1-OH) andGb3(d18:1/24:1)/GlcCer(d18:1/24:0) (Table 7);

and the one or more lipid-lipid concentration ratio(s) whose decrease(s)is (are) compared to the control is (are) selected from: CE 18:3/PC16:0/18:1, CE 18:0/SM (d18:1/14:0) (d18:1/13:1-OH), CE 14:0/PE18:0/18:2, CE 18:3/PC O-16:0/18:1-alkyl, CE 18:0/Gb3(d18:1/16:0) and CE18:0/Gb3(d18:1/24:1) (Table 7).

In yet another alternative embodiment, the present invention relates toa method for determining whether a subject not undergoing statintreatment who is suffering from type 2 diabetes mellitus is at risk todevelop one or more CVD complications, such as AMI and/or CVD death,comprising determining in a sample from said subject one or morelipid-clinical concentration ratio(s), wherein (an) increased ordecreased lipid-clinical concentration ratio(s) in said sample, whencompared to a control sample, is (are) indicative of said subject havingan increased risk of developing one or more CVD complications, such asAMI or CVD death, wherein the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: PC 18:1/18:1/lipoprotein(a), PCO-16:0/18:1-alkyl/lipoprotein(a), Gb3(d18:1/18:0)/lipoprotein(a), SM(d18:1/23:1) (d18:1/22:2-OH)/lipoprotein(a),Gb3(d18:1/24:1)/lipoprotein(a), Gb3(d18:1/16:0)/lipoprotein(a), PE18:0/18:2/lipoprotein(a), LacCer(d18:1/24:1)/lipoprotein(a),LacCer(d18:1/22:0)/lipoprotein(a), Gb3(d18:1/22:0)/lipoprotein(a), CE17:1/lipoprotein(a), Gb3(d18:1/24:0)/lipoprotein(a), PC16:0/18:2/lipoprotein(a), PC O-18:0/18:2-alkyl/lipoprotein(a),LacCer(d18:1/24:0)/lipoprotein(a), PC 17:0/18:2/lipoprotein(a), SM(d18:1/18:0)/lipoprotein(a), CE 15:0/lipoprotein(a), PCO-16:0/18:2-alkyl/lipoprotein(a), Gb3(d18:1/24:1)/LDL cholesterol,Gb3(d18:1/24:1)/apolipoprotein B, Gb3(d18:1/24:1)/total cholesterol,Gb3(d18:1/24:1)/apolipoprotein A-I, PC O-16:0/18:1-alkyl/LDLcholesterol, Gb3(d18:1/16:0)/LDL cholesterol, Gb3 (d18:1/24:1)/HDLcholesterol, Gb3 (d18:1/22:0)/LDL cholesterol, Gb3(d18:1/18:0)/LDLcholesterol, Gb3 (d18:1/24:0)/LDL cholesterol,Gb3(d18:1/16:0)/apolipoprotein B, PC O-16:0/18:1-alkyl/apolipoprotein B,PC O-16:0/18:1-alkyl/triglycerides, Gb3 (d18:1/16:0)/total cholesterol,Gb3(d18:1/22:0)/apolipoprotein B, PC 16:0/16:0/LDL cholesterol,Gb3(d18:1/18:0)/apolipoprotein B, PC 0-16:0/18:1-alkyl/totalcholesterol, SM (d18:1/24:1) (d18:1/23:2-OH)/LDL cholesterol,Gb3(d18:1/16:0)/triglycerides, PE 18:0/18:2/LDL cholesterol,Gb3(d18:1/22:0)/total cholesterol, PE 18:0/18:2/triglycerides,Gb3(d18:1/18:0)/total cholesterol, PE 18:0/18:2/total cholesterol, PC16:0/18:2/LDL cholesterol, PC 16:0/16:0/total cholesterol, PE18:0/18:2/apolipoprotein B and SM (d18:1/14:0) (d18:1/13:1-OH)/totalcholesterol

(Table 5c);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: CE14:0/HDL cholesterol and CE 14:0/supersensitive C-reactive protein(Table 5c).

In a preferred embodiment, the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: PC 18:1/18:1/lipoprotein(a),Gb3(d18:1/18:0)/lipoprotein(a), Gb3(d18:1/16:0)/lipoprotein(a) andLacCer(d18:1/24:1)/lipoprotein(a) (Table 7);

and the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is: CE 14:0/supersensitiveC-reactive protein (Table 7).

In another aspect, the present invention relates to a method forevaluating the effectiveness of a treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject not undergoingstatin treatment, said method comprising determining in a sample fromsaid subject one or more lipid-lipid concentration ratio(s), wherein(an) increased or decreased lipid-lipid concentration ratio(s) in saidsample, when compared to a control sample, is (are) indicative ofeffectiveness of said treatment, wherein the one or more lipid-lipidconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is (are) selected from: Gb3(d18:1/16:0)/PC 18:0/22:6,Cer(d18:1/18:0)/Cer(d18:1/24:0), Cer(d18:1/20:0)/Cer(d18:1/24:0), SM(d18:1/16:0) (d18:1/15:1-OH)/SM (d18:1/24:0) (d18:1/23:1-OH) andCer(d18:1/22:0)/Cer(d18:1/24:0) (Table 3);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/PC 16:0/18:2, SM (d18:1/24:0) (d18:1/23:1-OH)/SM(d18:1/24:1) (d18:1/23:2-OH), Cer(d18:1/24:0)/Cer(d18:1/24:1),Cer(d18:1/24:0)/SM (d18:1/14:0) (d18:1/13:1-OH),Cer(d18:1/24:0)/Gb3(d18:1/16:0) and Cer(d18:1/24:0)/SM (d18:1/16:1)(d18:1/15:2-OH) (Table 3).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: Cer(d18:1/24:0)/PC 16:0/18:2, SM (d18:1/24:0)(d18:1/23:1-OH)/SM (d18:1/24:1) (d18:1/23:2-OH), Cer(d18:1/24:0)/SM(d18:1/14:0) (d18:1/13:1-OH), Cer(d18:1/24:0)/Gb3(d18:1/16:0) andCer(d18:1/24:0)/SM (d18:1/16:1) (d18:1/15:2-OH) (Table 3).

In yet another alternative embodiment the present invention relates tomethod for evaluating the effectiveness of a treatment of CVD and/or oneor more of its complications, such as AMI or CVD death, in a subject notundergoing statin treatment, said method comprising determining in asample from said subject one or more lipid-clinical concentrationratio(s), wherein (an) increased or decreased lipid-clinicalconcentration ratio(s) in said sample, when compared to a controlsample, is (are) indicative of effectiveness of said treatment, whereinthe one or more lipid-clinical concentration ratio(s) whose increase(s)is (are) compared to the control is (are) selected from:GlcCer(d18:1/16:0)/total cholesterol, Gb3(d18:1/16:0)/apolipoprotein Gb3(d18:1/18:0)/apolipoprotein A-I, Gb3(d18:1/16:0)/HDL cholesterol and PC16:0/18:2/total cholesterol (Table 3);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/supersensitive C-reactive protein and PC18:0/22:6/supersensitive C-reactive protein (Table 3).

For the purposes of the invention, and particularly for lipid-clinicalconcentration ratios, an Apolipoprotein A-I measurement mayalternatively be an Apolipoprotein A-II measurement.

In another aspect the present invention relates to a method forevaluating the effectiveness of a treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject not undergoingstatin treatment and not suffering from type 2 diabetes mellitus saidmethod comprising determining in a sample from said subject theconcentration(s) of one or more lipid(s), wherein (an) increased ordecreased concentration(s) in said sample, when compared to a controlsample, is (are) indicative of effectiveness of said treatment, whereinthe one or more lipid(s) whose increase(s) in concentration is (are)compared to the control is (are) selected from: CE 19:1 oxCE 682.6,GlcCer(d18:1/16:0), SM (d18:1/18:1), CE 20:4, LacCer(d18:1/16:0), Cer18:1/16:0, SM 18:1/16:0 and CE 16:0 (Tables 4a, 6 and 8);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PC 18:0/22:6,SM (d18:1/23:1) (d18:1/22:2-OH), PC 16:0/22:6, SM 18:1/24:0 and LPC 16:0(Tables 4a, 6 and 8).

In one particular embodiment, the one or more lipid(s) whose increase(s)in concentration is (are) compared to the control is (are) selectedfrom: CE 19:1 (oxCE 682.6), CE 20:4, Cer 18:1/16:0 and SM 18:1/16:0(Tables 4a, 6 and 8);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PC 18:0/22:6and SM 18:1/24:0 and LPC 16:0 (Tables 4a, 6 and 8).

In a preferred embodiment, the one or more lipid(s) whose increase(s) inconcentration is (are) compared to the control is (are) selected from:GlcCer(d18:1/16:0), CE 20:4, LacCer(d18:1/16:0), Cer 18:1/16:0, SM18:1/16:0 and CE 16:0 (Table 8);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: PC 16:0/22:6, SM18:1/24:0 and LPC 16:0 (Table 8).

In one particularly preferred embodiment, the one or more lipid(s) whoseincrease(s) in concentration is (are) compared to the control is (are)selected from: CE 20:4, Cer 18:1/16:0 and SM 18:1/16:0 (Table 8); andthe one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: SM 18:1/24:0 and LPC16:0 (Table 8).

In an alternative embodiment, the present invention relates to a methodfor evaluating the effectiveness of a treatment of CVD and/or one ormore of its complications, such as AMI or CVD death, in a subject notundergoing statin treatment and not suffering from type 2 diabetesmellitus said method comprising determining in a sample from saidsubject one or more lipid-lipid concentration ratio(s), wherein (an)increased or decreased lipid-lipid concentration ratio(s) in saidsample, when compared to a control sample, is (are) indicative ofeffectiveness of said treatment, wherein the one or more lipid-lipidconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is (are) selected from: Cer(d18:1/18:0)/SM (d18:1/23:1)(d18:1/22:2-OH), Cer(d18:1/16:0)/PC 18:0/22:6, Cer(d18:1/18:0)/PC16:0/22:6, GlcCer(d18:1/16:0)/PC 18:0/22:6, GlcCer(d18:1/18:0)/SM(d18:1/23:1) (d18:1/22:2-OH), Cer(d18:1/16:0)/PC 16:0/22:6,Cer(d18:1/20:0)/SM (d18:1/23:1) (d18:1/22:2-OH), GlcCer(d18:1/18:0)/PC16:0/22:6, SM (d18:1/18:1)/SM (d18:1/23:1) (d18:1/22:2-OH),Cer(d18:1/20:0)/PC 16:0/22:6, SM (d18:1/16:0) (d18:1/15:1-OH)/SM(d18:1/23:1) (d18:1/22:2-OH), SM (d18:1/15:0) (d18:1/14:1-OH)/SM(d18:1/23:1) (d18:1/22:2-OH), GlcCer(d18:1/20:0)/SM (d18:1/23:1)(d18:1/22:2-OH), Cer(d18:1/24:1)/PC 18:0/22:6, SM (d18:1/16:1)(d18:1/15:2-OH)/SM (d18:1/23:1) (d18:1/22:2-OH), GlcCer(d18:1/16:0)/PC16:0/22:6, LacCer(d18:1/22:0)/PC 16:0/22:6, LacCer(d18:1/22:0)/SM(d18:1/23:1) (d18:1/22:2-OH), GlcCer(d18:1/20:0)/PC 16:0/22:6, CE16:0/PC 18:0/22:6, Cer(d18:1/22:0)/PC 18:0/22:6, CE 18:2/PC 18:0/22:6,CE 18:1/PC 18:0/22:6, Cer(d18:1/16:0)/Cer(d18:1/24:0),Gb3(d18:1/16:0)/PC 16:0/22:6, CE 20:4/PC 18:0/22:6, CE 22:6/PC18:0/22:6, PC 16:0/16:0/PC 16:0/22:6, CE 16:0/PC 16:0/22:6, CE 18:2/PC16:0/22:6, Gb3(d18:1/24:0)/PC 16:0/22:6, CE 18:1/PC 16:0/22:6, CE20:4/PC 16:0/22:6, SM (d18:1/16:1) (d18:1/15:2-OH)/SM (d18:1/24:0)(d18:1/23:1-OH), Cer(d18:1/16:0)/Cer(d18:1/22:0), CE16:0/Cer(d18:1/24:0), SM (d18:1/18:1)/SM (d18:1/24:0) (d18:1/23:1-OH),GlcCer(d18:1/18:0)/SM (d18:1/24:0) (d18:1/23:1-OH) and SM (d18:1/15:0)(d18:1/14:1-OH)/SM (d18:1/24:0) (d18:1/23:1-OH) (Table 4b);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/GlcCer(d18:1/18:0), Cer(d18:1/24:0)/GlcCer(d18:1/16:0),PC 16:0/22:6/SM (d18:1/16:0) (d18:1/15:1-OH), PC 18:0/22:6/SM(d18:1/14:0) (d18:1/13:1-OH), PC 16:0/22:6/SM (d18:1/16:1)(d18:1/15:2-OH), PC 18:0/22:6/SM (d18:1/16:0) (d18:1/15:1-OH), PC18:0/22:6/SM (d18:1/15:0) (d18:1/14:1-OH), PC 18:0/22:6/SM (d18:1/18:0),PC 18:0/22:6/SM (d18:1/16:1) (d18:1/15:2-OH) and PC 18:0/22:6/SM(d18:1/18:1) (Table 4b).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Cer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/22:2-OH),Cer(d18:1/16:0)/PC 18:0/22:6, Cer(d18:1/18:0)/PC 16:0/22:6,GlcCer(d18:1/16:0)/PC 18:0/22:6, GlcCer(d18:1/18:0)/SM (d18:1/23:1)(d18:1/22:2-OH), Cer(d18:1/16:0)/PC 16:0/22:6, Cer(d18:1/20:0)/SM(d18:1/23:1) (d18:1/22:2-OH), GlcCer(d18:1/18:0)/PC 16:0/22:6, SM(d18:1/18:1)/SM (d18:1/23:1) (d18:1/22:2-OH), Cer(d18:1/20:0)/PC16:0/22:6, SM (d18:1/16:0) (d18:1/15:1-OH)/SM (d18:1/23:1)(d18:1/22:2-OH), SM (d18:1/15:0) (d18:1/14:1-OH)/SM (d18:1/23:1)(d18:1/22:2-OH), GlcCer(d18:1/20:0)/SM (d18:1/23:1) (d18:1/22:2-OH),Cer(d18:1/24:1)/PC 18:0/22:6, SM (d18:1/16:1) (d18:1/15:2-OH)/SM(d18:1/23:1) (d18:1/22:2-OH), GlcCer(d18:1/16:0)/PC 16:0/22:6,LacCer(d18:1/22:0)/PC 16:0/22:6, CE 16:0/PC 18:0/22:6, CE 18:2/PC18:0/22:6 and Cer(d18:1/16:0)/Cer(d18:1/24:0) (Table 6);

and the one or more lipid-lipid concentration ratio(s) whose decrease(s)is (are) compared to the control is (are) selected from: PC 18:0/22:6/SM(d18:1/16:1) (d18:1/15:2-OH) and PC 18:0/22:6/SM (d18:1/18:1) (Table 6).

In yet another alternative embodiment, a method is provided forevaluating the effectiveness of a treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject not undergoingstatin treatment and not suffering from type 2 diabetes mellitus, saidmethod comprising determining in a sample from said subject one or morelipid-clinical concentration ratio(s), wherein (an) increased ordecreased lipid-clinical concentration ratio(s) in said sample, whencompared to a control sample, is (are) indicative of effectiveness ofsaid treatment, wherein the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Cer(d18:1/16:0)/HDL cholesterol,GlcCer(d18:1/16:0)/apolipoprotein A-I, CE 19:1 oxCE 682.6/apolipoproteinA-I, GlcCer(d18:1/18:0)/apolipoprotein A-I, GlcCer(d18:1/18:0)/HDLcholesterol, GlcCer(d18:1/16:0)/HDL cholesterol,GlcCer(d18:1/20:0)/apolipoprotein A-I, GlcCer(d18:1/18:0)/totalcholesterol and Cer(d18:1/16:0)/apolipoprotein B

(Table 4c);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:LacCer(d18:1/24:0)/supersensitive C-reactive protein, CE17:1/supersensitive C-reactive protein, SM (d18:1/16:0)(d18:1/15:1-OH)/supersensitive C-reactive protein,GlcCer(d18:1/24:0)/supersensitive C-reactive protein,Gb3(d18:1/22:0)/supersensitive C-reactive protein, PC18:0/20:3/apolipoprotein B, PC 16:0/16:1/supersensitive C-reactiveprotein, PC 16:0/22:6/total cholesterol, PC 16:0/22:6/apolipoprotein B,PC 18:1/18:1/supersensitive C-reactive protein, SM (d18:1/24:1)(d18:1/23:2-OH)/supersensitive C-reactive protein, PC18:0/18:2/supersensitive C-reactive protein,Cer(d18:1/24:1)/supersensitive C-reactive protein,Cer(d18:1/22:0)/supersensitive C-reactive protein, PC18:0/22:6/triglycerides, SM (d18:1/18:0)/supersensitive C-reactiveprotein, PC 18:0/20:3/supersensitive C-reactive protein, SM (d18:1/24:0)(d18:1/23:1-OH)/supersensitive C-reactive protein, PC16:0/18:1/supersensitive C-reactive protein, SM (d18:1/23:1)(d18:1/22:2-OH)/total cholesterol, PC 18:0/22:6/LDL cholesterol, PC18:0/22:6/total cholesterol, SM (d18:1/23:1) (d18:1/22:2-OH)/LDLcholesterol, SM (d18:1/23:1) (d18:1/22:2-OH)/apolipoprotein B, PC18:0/22:6/apolipoprotein B, SM (d18:1/23:0)(d18:1/22:1-OH)/supersensitive C-reactive protein, PC18:0/18:1/supersensitive C-reactive protein, LPC 16:0/supersensitiveC-reactive protein, PC 16:0/22:6/supersensitive C-reactive protein, SM(d18:1/23:1) (d18:1/22:2-OH)/supersensitive C-reactive protein, PC18:0/22:6/lipoprotein(a) and PC 16:0/18:0/supersensitive C-reactiveprotein (Table 4c).

In one particular embodiment, the one or more lipid-clinicalconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is (are) selected from: CE 19:1 oxCE 682.6/apolipoprotein A-I,GlcCer(d18:1/18:0)/HDL cholesterol, GlcCer(d18:1/16:0)/HDL cholesterol,GlcCer(d18:1/20:0)/apolipoprotein A-I and GlcCer(d18:1/18:0)/totalcholesterol (Table 4c).

In a preferred embodiment, the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is:Cer(d18:1/16:0)/HDL cholesterol (Table 6);

and/or the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: PC18:0/22:6/LDL cholesterol, PC 18:0/22:6/total cholesterol and PC18:0/22:6/apolipoprotein B (Table 6).

In another aspect, the present invention relates to a method forevaluating the effectiveness of a treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject not undergoingstatin treatment and suffering from type 2 diabetes mellitus said methodcomprising determining in a sample from said subject theconcentration(s) of one or more lipid(s), wherein (an) increased ordecreased concentration(s) in said sample, when compared to a controlsample, is (are) indicative of effectiveness of said treatment, whereinthe one or more lipid(s) whose increase(s) in concentration is (are)compared to the control is (are) selected from: Gb3(d18:1/24:1),Gb3(d18:1/16:0), GlcCer 18:1/16:0, LacCer 18:1/16:0 and PC 16:0/22:6(Tables 5a, 7 and 8);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PCO-16:0/20:4-alkyl, CE 20:4, CE 18:0, CE 14:0, CE 22:6, CE 18:3, GlcCer18:1/18:0 and SM 18:1/24:0 (Tables 5a, 7 and 8).

In one particular embodiment, the one or more lipid(s) whose increase(s)in concentration is (are) compared to the control is (are) selectedfrom: Gb3(d18:1/24:1), Gb3(d18:1/16:0), GlcCer 18:1/16:0 and LacCer18:1/16:0 (Tables 5a, 7 and 8);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PCO-16:0/20:4-alkyl, CE 20:4, CE 22:6, GlcCer 18:1/18:0 and SM 18:1/24:0(Tables 5a, 7 and 8).

In a preferred embodiment, the one or more lipid(s) whose increase(s) inconcentration is (are) compared to the control is (are) selected from:Gb3 18:1/16:0, GlcCer 18:1/16:0, LacCer 18:1/16:0 and PC 16:0/22:6(Table 8);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: CE 14:0, CE 22:6, CE18:3, GlcCer 18:1/18:0 and SM 18:1/24:0 (Table 8).

In one particularly preferred embodiment, the one or more lipid(s) whoseincrease(s) in concentration is (are) compared to the control is (are)selected from: Gb3 18:1/16:0, GlcCer 18:1/16:0 and LacCer 18:1/16:0(Table 8);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: CE 22:6, GlcCer18:1/18:0 and SM 18:1/24:0 (Table 8).

In an alternative embodiment, the present invention relates to a methodfor evaluating the effectiveness of a treatment of CVD and/or one ormore of its complications, such as AMI or CVD death, in a subject notundergoing statin treatment and suffering from type 2 diabetes mellitussaid method comprising determining in a sample from said subject one ormore lipid-lipid concentration ratio(s), wherein (an) increased ordecreased lipid-lipid concentration ratio(s) in said sample, whencompared to a control sample, is (are) indicative of effectiveness ofsaid treatment, wherein the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Gb3(d18:1/24:1)/SM (d18:1/17:0) (d18:1/16:1-OH),Gb3(d18:1/24:1)/PC O-16:0/20:4-alkyl, Gb3(d18:1/16:0)/SM (d18:1/17:0)(d18:1/16:1-OH), Gb3(d18:1/24:1)/GlcCer(d18:1/24:0),Gb3(d18:1/24:1)/GlcCer(d18:1/22:0) and Gb3(d18:1/22:0)/SM (d18:1/17:0)(d18:1/16:1-OH) (Table 5b);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: CE18:0/PC 18:0/18:2, PC 18:0/18:2/PE 18:0/18:2, Cer(d18:1/24:0)/PE18:0/18:2, CE 18:0/CE 18:1, CE 18:2/Gb3(d18:1/16:0), CE16:0/Gb3(d18:1/16:0), CE 18:0/PC 16:0/22:6, CE 14:0/Gb3(d18:1/24:0), CE18:0/SM (d18:1/16:0) (d18:1/15:1-OH), CE 18:3/SM (d18:1/14:0)(d18:1/13:1-OH), CE 18:0/PC 16:0/18:2, Cer(d18:1/24:0)/PCO-16:0/18:1-alkyl, CE 18:0/Gb3(d18:1/24:0), CE 18:3/PC 16:0/18:1, PC18:0/20:3/PC O-16:0/18:1-alkyl, CE 14:0/PC 16:0/16:0, CE17:1/Gb3(d18:1/16:0), CE 14:0/SM (d18:1/14:0) (d18:1/13:1-OH), PC18:0/20:3/PE 18:0/18:2, CE 18:0/SM (d18:1/14:0) (d18:1/13:1-OH), CE14:0/PC 16:0/18:1, CE 18:0/SM (d18:1/24:1) (d18:1/23:2-OH), CE18:0/Cer(d18:1/16:0), CE 18:0/Cer(d18:1/24:1), CE 18:0/PC 16:0/16:0, CE18:0/PC 18:1/18:1, CE 18:0/PC 16:0/18:1, CE 20:4/PC O-16:0/18:1-alkyl,CE 18:3/PE 18:0/18:2, CE 20:4/Gb3(d18:1/16:0), CE 14:0/PE 18:0/18:2, CE18:0/Cer(d18:1/26:1), CE 18:3/Gb3(d18:1/22:0), CE 14:0/Gb3(d18:1/16:0),CE 18:3/PC O-16:0/18:1-alkyl, CE 18:3/Gb3(d18:1/16:0), CE 14:0/PCO-16:0/18:1-alkyl, CE 20:4/Gb3(d18:1/18:0), CE 18:3/Gb3(d18:1/24:1), CE14:0/Gb3(d18:1/24:1), CE 20:5/PC O-16:0/18:1-alkyl, CE18:0/Gb3(d18:1/18:0), CE 18:0/Gb3(d18:1/16:0) and CE18:0/Gb3(d18:1/24:1) (Table 5b).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: CE 18:0/PC 18:0/18:2, PC 18:0/18:2/PE 18:0/18:2,Cer(d18:1/24:0)/PE 18:0/18:2, CE 18:0/CE 18:1, CE 18:2/Gb3(d18:1/16:0),CE 16:0/Gb3(d18:1/16:0), CE 18:0/PC 16:0/22:6, CE 14:0/Gb3(d18:1/24:0),CE 18:0/SM (d18:1/16:0) (d18:1/15:1-OH), CE 18:3/SM (d18:1/14:0)(d18:1/13:1-OH), CE 18:0/PC 16:0/18:2, Cer(d18:1/24:0)/PCO-16:0/18:1-alkyl, CE 18:0/Gb3(d18:1/24:0), CE 18:3/PC 16:0/18:1, PC18:0/20:3/PC O-16:0/18:1-alkyl, CE 14:0/PC 16:0/16:0, CE17:1/Gb3(d18:1/16:0), CE 14:0/SM (d18:1/14:0) (d18:1/13:1-OH), PC18:0/20:3/PE 18:0/18:2, CE 18:0/SM (d18:1/14:0) (d18:1/13:1-OH), CE14:0/PC 16:0/18:1, CE 18:0/SM (d18:1/24:1) (d18:1/23:2-OH), CE18:0/Cer(d18:1/24:1), CE 18:0/PC 16:0/16:0, CE 18:0/PC 18:1/18:1, CE18:0/PC 16:0/18:1, CE 20:4/PC O-16:0/18:1-alkyl, CE 18:3/PE 18:0/18:2,CE 20:4/Gb3(d18:1/16:0), CE 14:0/PE 18:0/18:2, CE 18:0/Cer(d18:1/26:1),CE 18:3/Gb3(d18:1/22:0), CE 14:0/Gb3(d18:1/16:0), CE 18:3/PC0-16:0/18:1-alkyl, CE 18:3/Gb3(d18:1/16:0), CE 14:0/PCO-16:0/18:1-alkyl, CE 20:4/Gb3(d18:1/18:0), CE 18:3/Gb3(d18:1/24:1), CE14:0/Gb3(d18:1/24:1), CE 20:5/PC O-16:0/18:1-alkyl, CE18:0/Gb3(d18:1/18:0), CE 18:0/Gb3(d18:1/16:0) and CE18:0/Gb3(d18:1/24:1) (Table 5b).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Gb3(d18:1/24:1)/SM (d18:1/17:0) (d18:1/16:1-OH),Gb3(d18:1/16:0)/SM (d18:1/17:0) (d18:1/16:1-OH) andGb3(d18:1/24:1)/GlcCer(d18:1/24:0) (Table 7); and the one or morelipid-lipid concentration ratio(s) whose decrease(s) is (are) comparedto the control is (are) selected from: CE 18:3/PC 16:0/18:1, CE 18:0/SM(d18:1/14:0) (d18:1/13:1-OH), CE 14:0/PE 18:0/18:2, CE 18:3/PCO-16:0/18:1-alkyl, CE 18:0/Gb3(d18:1/16:0) and CE 18:0/Gb3(d18:1/24:1)(Table 7).

In yet another alternative embodiment, a method is provided forevaluating the effectiveness of a treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject not undergoingstatin treatment and suffering from type 2 diabetes mellitus, saidmethod comprising determining in a sample from said subject one or morelipid-clinical concentration ratio(s), wherein (an) increased ordecreased lipid-clinical concentration ratio(s) in said sample, whencompared to a control sample, is (are) indicative of effectiveness ofsaid treatment, wherein the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: PC 18:1/18:1/lipoprotein(a), PCO-16:0/18:1-alkyl/lipoprotein(a), Gb3 (d18:1/18:0)/lipoprotein(a), SM(d18:1/23:1) (d18:1/22:2-OH)/lipoprotein(a),Gb3(d18:1/24:1)/lipoprotein(a), Gb3(d18:1/16:0)/lipoprotein(a), PE18:0/18:2/lipoprotein(a), LacCer(d18:1/24:1)/lipoprotein(a),LacCer(d18:1/22:0)/lipoprotein(a), Gb3 (d18:1/22:0)/lipoprotein(a), CE17:1/lipoprotein(a), Gb3(d18:1/24:0)/lipoprotein(a), PC16:0/18:2/lipoprotein(a), PC O-18:0/18:2-alkyl/lipoprotein(a),LacCer(d18:1/24:0)/lipoprotein(a), PC 17:0/18:2/lipoprotein(a), SM(d18:1/18:0)/lipoprotein(a), CE 15:0/lipoprotein(a), PCO-16:0/18:2-alkyl/lipoprotein(a), Gb3(d18:1/24:1)/LDL cholesterol,Gb3(d18:1/24:1)/apolipoprotein B, Gb3(d18:1/24:1)/total cholesterol,Gb3(d18:1/24:1)/apolipoprotein A-I, PC O-16:0/18:1-alkyl/LDLcholesterol, Gb3(d18:1/16:0)/LDL cholesterol, Gb3(d18:1/24:1)/HDLcholesterol, Gb3(d18:1/22:0)/LDL cholesterol, Gb3(d18:1/18:0)/LDLcholesterol, Gb3(d18:1/24:0)/LDL cholesterol,Gb3(d18:1/16:0)/apolipoprotein B, PC O-16:0/18:1-alkyl/apolipoprotein B,PC O-16:0/18:1-alkyl/triglycerides, Gb3(d18:1/16:0)/total cholesterol,Gb3(d18:1/22:0)/apolipoprotein B, PC 16:0/16:0/LDL cholesterol,Gb3(d18:1/18:0)/apolipoprotein B, PC O-16:0/18:1-alkyl/totalcholesterol, SM (d18:1/24:1) (d18:1/23:2-OH)/LDL cholesterol,Gb3(d18:1/16:0)/triglycerides, PE 18:0/18:2/LDL cholesterol,Gb3(d18:1/22:0)/total cholesterol, PE 18:0/18:2/triglycerides,Gb3(d18:1/18:0)/total cholesterol, PE 18:0/18:2/total cholesterol, PC16:0/18:2/LDL cholesterol, PC 16:0/16:0/total cholesterol, PE18:0/18:2/apolipoprotein B and SM (d18:1/14:0) (d18:1/13:1-OH)/totalcholesterol (Table 5c);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: CE14:0/HDL cholesterol and CE 14:0/supersensitive C-reactive protein(Table 5c).

In a preferred embodiment, the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: PC 18:1/18:1/lipoprotein(a),Gb3(d18:1/18:0)/lipoprotein(a), Gb3(d18:1/16:0)/lipoprotein(a) andLacCer(d18:1/24:1)/lipoprotein(a) (Table 7);

and the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is: CE 14:0/supersensitiveC-reactive protein (Table 7).

In yet another aspect the present invention relates to a method ofchoosing an appropriate treatment of CVD and/or one or more of itscomplications, such as AMI or CVD death, in a subject not undergoingstatin treatment, said method comprising determining in a sample fromsaid subject one or more lipid-lipid concentration ratio(s), wherein(an) increased or decreased lipid-lipid concentration ratio(s) in saidsample, when compared to a control sample, is (are) indicative of saidsubject being in need of treatment or a change in, or supplementationof, an already administered treatment, wherein the one or morelipid-lipid concentration ratio(s) whose increase(s) is (are) comparedto the control is (are) selected from: Gb3(d18:1/16:0)/PC 18:0/22:6,Cer(d18:1/18:0)/Cer(d18:1/24:0), Cer(d18:1/20:0)/Cer(d18:1/24:0), SM(d18:1/16:0) (d18:1/15:1-OH)/SM (d18:1/24:0) (d18:1/23:1-OH) andCer(d18:1/22:0)/Cer(d18:1/24:0) (Table 3);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/PC 16:0/18:2, SM (d18:1/24:0) (d18:1/23:1-OH)/SM(d18:1/24:1) (d18:1/23:2-OH), Cer(d18:1/24:0)/Cer(d18:1/24:1),Cer(d18:1/24:0)/SM (d18:1/14:0) (d18:1/13:1-OH),Cer(d18:1/24:0)/Gb3(d18:1/16:0) and Cer(d18:1/24:0)/SM (d18:1/16:1)(d18:1/15:2-OH) (Table 3).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: Cer(d18:1/24:0)/PC 16:0/18:2, SM (d18:1/24:0)(d18:1/23:1-OH)/SM (d18:1/24:1) (d18:1/23:2-OH), Cer(d18:1/24:0)/SM(d18:1/14:0) (d18:1/13:1-OH), Cer(d18:1/24:0)/Gb3(d18:1/16:0) andCer(d18:1/24:0)/SM (d18:1/16:1) (d18:1/15:2-OH) (Table 3).

In yet another alternative embodiment, the present invention relates toa method of choosing an appropriate treatment of CVD and/or one or moreof its complications, such as AMI or CVD death, in a subject notundergoing statin treatment, said method comprising determining in asample from said subject one or more lipid-clinical concentrationratio(s), wherein (an) increased or decreased lipid-clinicalconcentration ratio(s) in said sample, when compared to a controlsample, is (are) indicative of said subject being in need of treatmentor a change in, or supplementation of, an already administeredtreatment, wherein the one or more lipid-clinical concentration ratio(s)whose increase(s) is (are) compared to the control is (are) selectedfrom: GlcCer(d18:1/16:0)/total cholesterol,Gb3(d18:1/16:0)/apolipoprotein A-I, Gb3(d18:1/18:0)/apolipoprotein A-I,Gb3(d18:1/16:0)/HDL cholesterol and PC 16:0/18:2/total cholesterol(Table 3);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/supersensitive C-reactive protein and PC18:0/22:6/supersensitive C-reactive protein (Table 3).

For the purposes of the invention, and particularly for lipid-clinicalconcentration ratios, an Apolipoprotein A-I measurement mayalternatively be an Apolipoprotein A-II measurement.

In a further aspect, the invention relates to a method of choosing anappropriate treatment of CVD and/or one or more of its complications,such as AMI or CVD death, in a subject not undergoing statin treatmentand not suffering from type 2 diabetes mellitus, said method comprisingdetermining in a sample from said subject the concentration(s) of one ormore lipid(s), wherein (an) increased or decreased concentration(s) insaid sample, when compared to a control sample, is (are) indicative ofsaid subject being in need of treatment or a change in, orsupplementation of, an already administered treatment, wherein the oneor more lipid(s) whose increase(s) in concentration is (are) compared tothe control is (are) selected from: CE 19:1 (oxCE 682.6),GlcCer(d18:1/16:0), SM (d18:1/18:1), CE 20:4, LacCer(d18:1/16:0), Cer18:1/16:0, SM 18:1/16:0 and CE 16:0 (Tables 4a, 6 and 8);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PC 18:0/22:6,SM (d18:1/23:1) (d18:1/22:2-OH), PC 16:0/22:6, SM 18:1/24:0 and LPC 16:0(Tables 4a, 6 and 8).

In one particular embodiment, the one or more lipid(s) whose increase(s)in concentration is (are) compared to the control is (are) selectedfrom: CE 19:1 (oxCE 682.6), CE 20:4, Cer 18:1/16:0 and SM 18:1/16:0(Tables 4a, 6 and 8);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PC 18:0/22:6,SM 18:1/24:0 and LPC 16:0 (Tables 4a, 6 and 8).

In a preferred embodiment, the one or more lipid(s) whose increase(s) inconcentration is (are) compared to the control is (are) selected from:GlcCer(d18:1/16:0), CE 20:4, LacCer(d18:1/16:0), Cer 18:1/16:0, SM18:1/16:0 and CE 16:0 (Table 8);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: PC 16:0/22:6, SM18:1/24:0 and LPC 16:0 (Table 8).

In one particularly preferred embodiment, the one or more lipid(s) whoseincrease(s) in concentration is (are) compared to the control is (are)selected from: CE 20:4, Cer 18:1/16:0 and SM 18:1/16:0 (Table 8); andthe one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: SM 18:1/24:0 and LPC16:0 (Table 8).

In an alternative embodiment, the present invention relates to a methodof choosing an appropriate treatment of CVD and/or one or more of itscomplications, such as AMI or CVD death, in a subject not undergoingstatin treatment and not suffering from type 2 diabetes mellitus, saidmethod comprising determining in a sample from said subject one or morelipid-lipid concentration ratio(s), wherein (an) increased or decreasedlipid-lipid concentration ratio(s) in said sample, when compared to acontrol sample, is (are) indicative of said subject being in need oftreatment or a change in, or supplementation of, an already administeredtreatment, wherein the one or more lipid-lipid concentration ratio(s)whose increase(s) is (are) compared to the control is (are) selectedfrom: Cer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/22:2-OH),Cer(d18:1/16:0)/PC 18:0/22:6, Cer(d18:1/18:0)/PC 16:0/22:6,GlcCer(d18:1/16:0)/PC 18:0/22:6, GlcCer(d18:1/18:0)/SM (d18:1/23:1)(d18:1/22:2-OH), Cer(d18:1/16:0)/PC 16:0/22:6, Cer(d18:1/20:0)/SM(d18:1/23:1) (d18:1/22:2-OH), GlcCer(d18:1/18:0)/PC 16:0/22:6, SM(d18:1/18:1)/SM (d18:1/23:1) (d18:1/22:2-OH), Cer(d18:1/20:0)/PC16:0/22:6, SM (d18:1/16:0) (d18:1/15:1-OH)/SM (d18:1/23:1)(d18:1/22:2-OH), SM (d18:1/15:0) (d18:1/14:1-OH)/SM (d18:1/23:1)(d18:1/22:2-OH), GlcCer(d18:1/20:0)/SM (d18:1/23:1) (d18:1/22:2-OH),Cer(d18:1/24:1)/PC 18:0/22:6, SM (d18:1/16:1) (d18:1/15:2-OH)/SM(d18:1/23:1) (d18:1/22:2-OH), GlcCer(d18:1/16:0)/PC 16:0/22:6,LacCer(d18:1/22:0)/PC 16:0/22:6, LacCer(d18:1/22:0)/SM (d18:1/23:1)(d18:1/22:2-OH), GlcCer(d18:1/20:0)/PC 16:0/22:6, CE 16:0/PC 18:0/22:6,Cer(d18:1/22:0)/PC 18:0/22:6, CE 18:2/PC 18:0/22:6, CE 18:1/PC18:0/22:6, Cer(d18:1/16:0)/Cer(d18:1/24:0), Gb3(d18:1/16:0)/PC16:0/22:6, CE 20:4/PC 18:0/22:6, CE 22:6/PC 18:0/22:6, PC 16:0/16:0/PC16:0/22:6, CE 16:0/PC 16:0/22:6, CE 18:2/PC 16:0/22:6,Gb3(d18:1/24:0)/PC 16:0/22:6, CE 18:1/PC 16:0/22:6, CE 20:4/PC16:0/22:6, SM (d18:1/16:1) (d18:1/15:2-OH)/SM (d18:1/24:0)(d18:1/23:1-OH), Cer(d18:1/16:0)/Cer(d18:1/22:0), CE16:0/Cer(d18:1/24:0), SM (d18:1/18:1)/SM (d18:1/24:0) (d18:1/23:1-OH),GlcCer(d18:1/18:0)/SM (d18:1/24:0) (d18:1/23:1-OH) and SM (d18:1/15:0)(d18:1/14:1-OH)/SM (d18:1/24:0) (d18:1/23:1-OH) (Table 4b);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/GlcCer(d18:1/18:0), Cer(d18:1/24:0)/GlcCer(d18:1/16:0),PC 16:0/22:6/SM (d18:1/16:0) (d18:1/15:1-OH), PC 18:0/22:6/SM(d18:1/14:0) (d18:1/13:1-OH), PC 16:0/22:6/SM (d18:1/16:1)(d18:1/15:2-OH), PC 18:0/22:6/SM (d18:1/16:0) (d18:1/15:1-OH), PC18:0/22:6/SM (d18:1/15:0) (d18:1/14:1-OH), PC 18:0/22:6/SM (d18:1/18:0),PC 18:0/22:6/SM (d18:1/16:1) (d18:1/15:2-OH) and PC 18:0/22:6/SM(d18:1/18:1) (Table 4b).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Cer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/22:2-OH),Cer(d18:1/16:0)/PC 18:0/22:6, Cer(d18:1/18:0)/PC 16:0/22:6,GlcCer(d18:1/16:0)/PC 18:0/22:6, GlcCer(d18:1/18:0)/SM (d18:1/23:1)(d18:1/22:2-OH), Cer(d18:1/16:0)/PC 16:0/22:6, Cer(d18:1/20:0)/SM(d18:1/23:1) (d18:1/22:2-OH), GlcCer(d18:1/18:0)/PC 16:0/22:6, SM(d18:1/18:1)/SM (d18:1/23:1) (d18:1/22:2-OH), Cer(d18:1/20:0)/PC16:0/22:6, SM (d18:1/16:0) (d18:1/15:1-OH)/SM (d18:1/23:1)(d18:1/22:2-OH), SM (d18:1/15:0) (d18:1/14:1-OH)/SM (d18:1/23:1)(d18:1/22:2-OH), GlcCer(d18:1/20:0)/SM (d18:1/23:1) (d18:1/22:2-OH),Cer(d18:1/24:1)/PC 18:0/22:6, SM (d18:1/16:1) (d18:1/15:2-OH)/SM(d18:1/23:1) (d18:1/22:2-OH), GlcCer(d18:1/16:0)/PC 16:0/22:6,LacCer(d18:1/22:0)/PC 16:0/22:6, CE 16:0/PC 18:0/22:6, CE 18:2/PC18:0/22:6 and Cer(d18:1/16:0)/Cer(d18:1/24:0) (Table 6);

and the one or more lipid-lipid concentration ratio(s) whose decrease(s)is (are) compared to the control is (are) selected from: PC 18:0/22:6/SM(d18:1/16:1) (d18:1/15:2-OH) and PC 18:0/22:6/SM (d18:1/18:1) (Table 6).

In yet another alternative embodiment the present invention relates to amethod of choosing an appropriate treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject not undergoingstatin treatment and not suffering from type 2 diabetes mellitus, saidmethod comprising determining in a sample from said subject one or morelipid-clinical concentration ratio(s), wherein (an) increased ordecreased lipid-clinical concentration ratio(s) in said sample, whencompared to a control sample, is (are) indicative of said subject beingin need of treatment or a change in, or supplementation of, an alreadyadministered treatment, wherein the one or more lipid-clinicalconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is (are) selected from: Cer(d18:1/16:0)/HDL cholesterol,GlcCer(d18:1/16:0)/apolipoprotein A-I, CE 19:1 oxCE 682.6/apolipoproteinA-I, GlcCer(d18:1/18:0)/apolipoprotein A-I, GlcCer(d18:1/18:0)/HDLcholesterol, GlcCer(d18:1/16:0)/HDL cholesterol,GlcCer(d18:1/20:0)/apolipoprotein A-I, GlcCer(d18:1/18:0)/totalcholesterol and Cer(d18:1/16:0)/apolipoprotein B (Table 4c);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:LacCer(d18:1/24:0)/supersensitive C-reactive protein, CE17:1/supersensitive C-reactive protein, SM (d18:1/16:0)(d18:1/15:1-OH)/supersensitive C-reactive protein,GlcCer(d18:1/24:0)/supersensitive C-reactive protein,Gb3(d18:1/22:0)/supersensitive C-reactive protein, PC18:0/20:3/apolipoprotein B, PC 16:0/16:1/supersensitive C-reactiveprotein, PC 16:0/22:6/total cholesterol, PC 16:0/22:6/apolipoprotein B,PC 18:1/18:1/supersensitive C-reactive protein, SM (d18:1/24:1)(d18:1/23:2-OH)/supersensitive C-reactive protein, PC18:0/18:2/supersensitive C-reactive protein,Cer(d18:1/24:1)/supersensitive C-reactive protein,Cer(d18:1/22:0)/supersensitive C-reactive protein, PC18:0/22:6/triglycerides, SM (d18:1/18:0)/supersensitive C-reactiveprotein, PC 18:0/20:3/supersensitive C-reactive protein, SM (d18:1/24:0)(d18:1/23:1-OH)/supersensitive C-reactive protein, PC16:0/18:1/supersensitive C-reactive protein, SM (d18:1/23:1)(d18:1/22:2-OH)/total cholesterol, PC 18:0/22:6/LDL cholesterol, PC18:0/22:6/total cholesterol, SM (d18:1/23:1) (d18:1/22:2-OH)/LDLcholesterol, SM (d18:1/23:1) (d18:1/22:2-OH)/apolipoprotein B, PC18:0/22:6/apolipoprotein B, SM (d18:1/23:0)(d18:1/22:1-OH)/supersensitive C-reactive protein, PC18:0/18:1/supersensitive C-reactive protein, LPC 16:0/supersensitiveC-reactive protein, PC 16:0/22:6/supersensitive C-reactive protein, SM(d18:1/23:1) (d18:1/22:2-OH)/supersensitive C-reactive protein, PC18:0/22:6/lipoprotein(a) and PC 16:0/18:0/supersensitive C-reactiveprotein (Table 4c).

In one particular embodiment, the one or more lipid-clinicalconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is (are) selected from: CE 19:1 (oxCE 682.6)/apolipoprotein A-I,GlcCer(d18:1/18:0)/HDL cholesterol, GlcCer(d18:1/16:0)/HDL cholesterol,GlcCer(d18:1/20:0)/apolipoprotein A-I and GlcCer(d18:1/18:0)/totalcholesterol (Table 4c).

In a preferred embodiment, the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is:Cer(d18:1/16:0)/HDL cholesterol (Table 6);

and/or the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: PC18:0/22:6/LDL cholesterol, PC 18:0/22:6/total cholesterol and PC18:0/22:6/apolipoprotein B (Table 6).

In another aspect, the present invention relates a method of choosing anappropriate treatment of CVD and/or one or more of its complications,such as AMI or CVD death, in a subject not undergoing statin treatmentwho is suffering from type 2 diabetes mellitus, said method comprisingdetermining in a sample from said subject the concentration(s) of one ormore lipid(s), wherein (an) increased or decreased concentration(s) insaid sample, when compared to a control sample, is (are) indicative ofsaid subject being in need of treatment or a change in, orsupplementation of, an already administered treatment, wherein the oneor more lipid(s) whose increase(s) in concentration is (are) compared tothe control is (are) selected from: Gb3(d18:1/24:1), Gb3(d18:1/16:0),GlcCer 18:1/16:0, LacCer 18:1/16:0 and PC 16:0/22:6 (Tables 5a, 7 and8);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PCO-16:0/20:4-alkyl, CE 20:4, CE 18:0, CE 14:0, CE 22:6, CE 18:3, GlcCer18:1/18:0 and SM 18:1/24:0 (Tables 5a, 7 and 8).

In one particular embodiment, the one or more lipid(s) whose increase(s)in concentration is (are) compared to the control is (are) selectedfrom: Gb3(d18:1/24:1), Gb3(d18:1/16:0), GlcCer 18:1/16:0 and LacCer18:1/16:0 (Tables 5a, 7 and 8);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PCO-16:0/20:4-alkyl, CE 20:4, CE 22:6, GlcCer 18:1/18:0 and SM 18:1/24:0(Tables 5a, 7 and 8).

In a preferred embodiment, the one or more lipid(s) whose increase(s) inconcentration is (are) compared to the control is (are) selected from:Gb3 18:1/16:0, GlcCer 18:1/16:0, LacCer 18:1/16:0 and PC 16:0/22:6(Table 8);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: CE 14:0, CE 22:6, CE18:3, GlcCer 18:1/18:0 and SM 18:1/24:0 (Table 8).

In one particularly preferred embodiment, the one or more lipid(s) whoseincrease(s) in concentration is (are) compared to the control is (are)selected from: Gb3 18:1/16:0, GlcCer 18:1/16:0 and LacCer 18:1/16:0(Table 8);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: CE 22:6, GlcCer18:1/18:0 and SM 18:1/24:0 (Table 8).

In an alternative embodiment, the present invention relates to a methodof choosing an appropriate treatment of CVD and/or one or more of itscomplications, such as AMI or CVD death, in a subject not undergoingstatin treatment who is suffering from type 2 diabetes mellitus, saidmethod comprising determining in a sample from said subject one or morelipid-lipid concentration ratio(s), wherein (an) increased or decreasedlipid-lipid concentration ratio(s) in said sample, when compared to acontrol sample, is (are) indicative of said subject being in need oftreatment or a change in, or supplementation of, an already administeredtreatment, wherein the one or more lipid-lipid concentration ratio(s)whose increase(s) is (are) compared to the control is (are) selectedfrom: Gb3(d18:1/24:1)/SM (d18:1/17:0) (d18:1/16:1-OH),Gb3(d18:1/24:1)/PC O-16:0/20:4-alkyl, Gb3(d18:1/16:0)/SM (d18:1/17:0)(d18:1/16:1-OH), Gb3(d18:1/24:1)/GlcCer(d18:1/24:0),Gb3(d18:1/24:1)/GlcCer(d18:1/22:0) and Gb3(d18:1/22:0)/SM (d18:1/17:0)(d18:1/16:1-OH) (Table 5b);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: CE18:0/PC 18:0/18:2, PC 18:0/18:2/PE 18:0/18:2, Cer(d18:1/24:0)/PE18:0/18:2, CE 18:0/CE 18:1, CE 18:2/Gb3(d18:1/16:0), CE16:0/Gb3(d18:1/16:0), CE 18:0/PC 16:0/22:6, CE 14:0/Gb3(d18:1/24:0), CE18:0/SM (d18:1/16:0) (d18:1/15:1-OH), CE 18:3/SM (d18:1/14:0)(d18:1/13:1-OH), CE 18:0/PC 16:0/18:2, Cer(d18:1/24:0)/PCO-16:0/18:1-alkyl, CE 18:0/Gb3(d18:1/24:0), CE 18:3/PC 16:0/18:1, PC18:0/20:3/PC O-16:0/18:1-alkyl, CE 14:0/PC 16:0/16:0, CE17:1/Gb3(d18:1/16:0), CE 14:0/SM (d18:1/14:0) (d18:1/13:1-OH), PC18:0/20:3/PE 18:0/18:2, CE 18:0/SM (d18:1/14:0) (d18:1/13:1-OH), CE14:0/PC 16:0/18:1, CE 18:0/SM (d18:1/24:1) (d18:1/23:2-OH), CE18:0/Cer(d18:1/16:0), CE 18:0/Cer(d18:1/24:1), CE 18:0/PC 16:0/16:0, CE18:0/PC 18:1/18:1, CE 18:0/PC 16:0/18:1, CE 20:4/PC O-16:0/18:1-alkyl,CE 18:3/PE 18:0/18:2, CE 20:4/Gb3(d18:1/16:0), CE 14:0/PE 18:0/18:2, CE18:0/Cer(d18:1/26:1), CE 18:3/Gb3(d18:1/22:0), CE 14:0/Gb3(d18:1/16:0),CE 18:3/PC O-16:0/18:1-alkyl, CE 18:3/Gb3(d18:1/16:0), CE 14:0/PCO-16:0/18:1-alkyl, CE 20:4/Gb3(d18:1/18:0), CE 18:3/Gb3(d18:1/24:1), CE14:0/Gb3(d18:1/24:1), CE 20:5/PC O-16:0/18:1-alkyl, CE18:0/Gb3(d18:1/18:0), CE 18:0/Gb3(d18:1/16:0) and CE18:0/Gb3(d18:1/24:1) (Table 5b).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: CE 18:0/PC 18:0/18:2, PC 18:0/18:2/PE 18:0/18:2,Cer(d18:1/24:0)/PE 18:0/18:2, CE 18:0/CE 18:1, CE 18:2/Gb3(d18:1/16:0),CE 16:0/Gb3(d18:1/16:0), CE 18:0/PC 16:0/22:6, CE 14:0/Gb3(d18:1/24:0),CE 18:0/SM (d18:1/16:0) (d18:1/15:1-OH), CE 18:3/SM (d18:1/14:0)(d18:1/13:1-OH), CE 18:0/PC 16:0/18:2, Cer(d18:1/24:0)/PCO-16:0/18:1-alkyl, CE 18:0/Gb3(d18:1/24:0), CE 18:3/PC 16:0/18:1, PC18:0/20:3/PC O-16:0/18:1-alkyl, CE 14:0/PC 16:0/16:0, CE17:1/Gb3(d18:1/16:0), CE 14:0/SM (d18:1/14:0) (d18:1/13:1-OH), PC18:0/20:3/PE 18:0/18:2, CE 18:0/SM (d18:1/14:0) (d18:1/13:1-OH), CE14:0/PC 16:0/18:1, CE 18:0/SM (d18:1/24:1) (d18:1/23:2-OH), CE18:0/Cer(d18:1/24:1), CE 18:0/PC 16:0/16:0, CE 18:0/PC 18:1/18:1, CE18:0/PC 16:0/18:1, CE 20:4/PC O-16:0/18:1-alkyl, CE 18:3/PE 18:0/18:2,CE 20:4/Gb3(d18:1/16:0), CE 14:0/PE 18:0/18:2, CE 18:0/Cer(d18:1/26:1),CE 18:3/Gb3(d18:1/22:0), CE 14:0/Gb3(d18:1/16:0), CE 18:3/PC0-16:0/18:1-alkyl, CE 18:3/Gb3(d18:1/16:0), CE 14:0/PCO-16:0/18:1-alkyl, CE 20:4/Gb3(d18:1/18:0), CE 18:3/Gb3(d18:1/24:1), CE14:0/Gb3(d18:1/24:1), CE 20:5/PC O-16:0/18:1-alkyl, CE18:0/Gb3(d18:1/18:0), CE 18:0/Gb3(d18:1/16:0) and CE18:0/Gb3(d18:1/24:1) (Table 5b).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Gb3(d18:1/24:1)/SM (d18:1/17:0) (d18:1/16:1-OH),Gb3(d18:1/16:0)/SM (d18:1/17:0) (d18:1/16:1-OH) andGb3(d18:1/24:1)/GlcCer(d18:1/24:0) (Table 7); and the one or morelipid-lipid concentration ratio(s) whose decrease(s) is (are) comparedto the control is (are) selected from: CE 18:3/PC 16:0/18:1, CE 18:0/SM(d18:1/14:0) (d18:1/13:1-OH), CE 14:0/PE 18:0/18:2, CE 18:3/PCO-16:0/18:1-alkyl, CE 18:0/Gb3(d18:1/16:0) and CE 18:0/Gb3(d18:1/24:1)(Table 7).

In yet another alternative embodiment the present invention relates to amethod of choosing an appropriate treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject not undergoingstatin treatment who is suffering from type 2 diabetes mellitus, saidmethod comprising determining in a sample from said subject one or morelipid-clinical concentration ratio(s), wherein (an) increased ordecreased lipid-clinical concentration ratio(s) in said sample, whencompared to a control sample is (are) indicative of said subject beingin need of treatment or a change in, or supplementation of, an alreadyadministered treatment, wherein the one or more lipid-clinicalconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is (are) selected from: PC 18:1/18:1/lipoprotein(a), PCO-16:0/18:1-alkyl/lipoprotein(a), Gb3 (d18:1/18:0)/lipoprotein(a), SM(d18:1/23:1) (d18:1/22:2-OH)/lipoprotein(a),Gb3(d18:1/24:1)/lipoprotein(a), Gb3(d18:1/16:0)/lipoprotein(a), PE18:0/18:2/lipoprotein(a), LacCer(d18:1/24:1)/lipoprotein(a),LacCer(d18:1/22:0)/lipoprotein(a), Gb3 (d18:1/22:0)/lipoprotein(a), CE17:1/lipoprotein(a), Gb3(d18:1/24:0)/lipoprotein(a), PC16:0/18:2/lipoprotein(a), PC O-18:0/18:2-alkyl/lipoprotein(a),LacCer(d18:1/24:0)/lipoprotein(a), PC 17:0/18:2/lipoprotein(a), SM(d18:1/18:0)/lipoprotein(a), CE 15:0/lipoprotein(a), PCO-16:0/18:2-alkyl/lipoprotein(a), Gb3(d18:1/24:1)/LDL cholesterol,Gb3(d18:1/24:1)/apolipoprotein B, Gb3 (d18:1/24:1)/total cholesterol,Gb3(d18:1/24:1)/apolipoprotein A-I, PC O-16:0/18:1-alkyl/LDLcholesterol, Gb3(d18:1/16:0)/LDL cholesterol, Gb3(d18:1/24:1)/HDLcholesterol, Gb3(d18:1/22:0)/LDL cholesterol, Gb3 (d18:1/18:0)/LDLcholesterol, Gb3(d18:1/24:0)/LDL cholesterol,Gb3(d18:1/16:0)/apolipoprotein B, PC O-16:0/18:1-alkyl/apolipoprotein B,PC O-16:0/18:1-alkyl/triglycerides, Gb3 (d18:1/16:0)/total cholesterol,Gb3(d18:1/22:0)/apolipoprotein B, PC 16:0/16:0/LDL cholesterol, Gb3(d18:1/18:0)/apolipoprotein B, PC O-16:0/18:1-alkyl/total cholesterol,SM (d18:1/24:1) (d18:1/23:2-OH)/LDL cholesterol,Gb3(d18:1/16:0)/triglycerides, PE 18:0/18:2/LDL cholesterol, Gb3(d18:1/22:0)/total cholesterol, PE 18:0/18:2/triglycerides,Gb3(d18:1/18:0)/total cholesterol, PE 18:0/18:2/total cholesterol, PC16:0/18:2/LDL cholesterol, PC 16:0/16:0/total cholesterol, PE18:0/18:2/apolipoprotein B and SM (d18:1/14:0) (d18:1/13:1-OH)/totalcholesterol (Table 5c);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: CE14:0/HDL cholesterol and CE 14:0/supersensitive C-reactive protein(Table 5c).

In a preferred embodiment, the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: PC 18:1/18:1/lipoprotein(a),Gb3(d18:1/18:0)/lipoprotein(a), Gb3(d18:1/16:0)/lipoprotein(a) andLacCer(d18:1/24:1)/lipoprotein(a) (Table 7);

and the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is: CE 14:0/supersensitiveC-reactive protein (Table 7).

In connection with all aspects and embodiments of the inventiondescribed and claimed herein, the determination of the lipidconcentration(s), the lipid-lipid concentration ratio(s) or thelipid-clinical concentration ratio(s) is typically performed using anassay.

In one embodiment of the invention, in which the treatment effectivenessis to be evaluated or the treatment is to be chosen as appropriate inaccordance with the methods described and claimed herein, said treatmentis a lipid modifying treatment.

The invention further encompasses the analysis of lipid concentrations,lipid-lipid concentration ratios and/or lipid-clinical concentrationratios in samples from a subject that has been previously treated withone or more statins and/or any other HMG-CoA reductase inhibitor.

For the purposes of the invention, at least one lipid concentration,lipid-lipid concentration ratio or lipid-clinical concentration ratiofrom Tables 3-8, or combinations thereof, may be determined to assesswhether the patient is at risk to develop one or more of CVDcomplications, such as AMI or CVD death; to evaluate the effectivenessof the treatment of CVD and/or one or more of its complications, such asAMI or CVD death in a subject; or to choose an appropriate treatment ofCVD and/or one or more of its complications, such as AMI or CVD death ina subject. However, it is also possible, and may be advantageous, todetermine at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, or at least 8 lipid concentrations, lipid-lipid concentrationratios or lipid-clinical concentration ratios from Tables 3-8, orcombinations thereof, in this regard. Where more than one lipidomicmarkers are determined and used for the assessment, it may beadvantageous that a specific lipid concentration, lipid-lipidconcentration ratio, lipid-clinical concentration ratio or combinationthereof, is given greater weight than others in the above-mentionedassessment, evaluation or choice.

In the context of the present invention, CVD is typically characterizedby coronary artery disease, peripheral artery disease, a stroke and/orCVD death. The CVD in the subject whose sample is analyzed in accordancewith the invention may be atherosclerosis-induced. Generally, theinvention embodies methods involving subjects who are at risk ofdeveloping CVD, and have atherosclerosis. Alternatively, the inventionembodies methods involving subjects who are at risk of developing CVD,and do not have atherosclerosis.

In a further embodiment, the methods of the invention may furthercomprise determining the serum or plasma level of total cholesterol,low-density lipoprotein cholesterol (LDL-C), high-density lipoproteincholesterol (HDL-C), Apolipoprotein B (ApoB) and/or Apolipoprotein C-IIIin the subject's sample. In one embodiment of the invention, the subjectdoes not have elevated serum or plasma levels of one or more of totalcholesterol, low-density lipoprotein cholesterol (LDL-C), ApolipoproteinC-III or Apolipoprotein B (ApoB), or a decreased serum level ofHDL-cholesterol (HDL-C).

In accordance with all aspects and embodiments described and claimedherein, both the sample from the subject and the control sample ispreferably a blood sample, more preferably a blood plasma sample, oralso preferably a blood serum sample. It may also be urine or tissue ora fraction of blood, blood plasma, blood serum, urine or tissue e.g., alipoprotein fraction. A blood sample can be prepared and plasma orserum, or fractions thereof, can be separated therefrom with techniqueswell known to the person skilled in the art. Alternatively, both thesample from the subject and the control sample may also be a tissuesample, e.g., artery tissue, such as carotid artery tissue, or arteryplaque material, such as carotid artery plaque material.

Collecting information on a lipidomic marker (i.e., the concentration(s)of (a) lipid(s), lipid-lipid concentration ratio(s), or lipid-clinicalconcentration ratio(s) or combinations thereof, i.e., correspondingprofile(s)) from the sample of a patient and, where appropriate, acorresponding control sample, can be performed with various chemical andhigh-resolution analytical techniques. Suitable analytical techniquesinclude, but are not limited to, mass spectrometry and nuclear resonancespectroscopy. Any high-resolution technique capable of resolvingindividual lipids or lipid classes and providing structural informationof the same can be used to collect the information on the lipidomicmarker in question, e.g., lipid profile from the biological sample. Formethods of the present invention the level of the lipid is determined byusing mass spectrometry, nuclear magnetic resonance spectroscopy,fluorescence spectroscopy or dual polarisation interferometry, a highperformance separation method such as HPLC, UHPLC or UPLC and/or animmunoassay such as an ELISA. According to an alternative or furtherembodiment an analyte in a sample can be detected and/or quantified bycombining the analyte with a binding moiety capable of specificallybinding the analyte. The binding moiety can include, for example, amember of a ligand-receptor pair, i.e., a pair of molecules capable ofhaving a specific binding interaction. The binding moiety can alsoinclude, for example, a member of a specific binding pair, such asantibody-antigen, enzyme-substrate, nucleic acid-based ligands, otherprotein ligands, or other specific binding pairs known in the art. In apreferred embodiment, the lipidomic profile is collected with massspectrometry (MS), wherein the MS instrument may be coupled to directinfusion methods and high performance separation methods such as HPLC,UHPLC or UPLC. The amount of the individual lipids or lipid classes inthe collected lipidomic profile is used when comparing the collectedlipid profile to a control.

Collecting the information on the lipidomic marker with massspectrometry (MS) is one of the preferred embodiments of the currentinvention. The MS instrument can be coupled to a direct sample infusionmethod, such as a robotic nanoflow ion source device, or to a highperformance separation method such as high performance liquidchromatography (HPLC), ultra high pressure liquid chromatography (UHPLC)or ultra performance liquid chromatography (UPLC).

The methods of the present invention may be used for determining a riskof said patient to develop CVD complications, particularly severe CVDcomplications such as death and myocardial infarction (MI), includingacute myocardial infarction (AMI).

In one embodiment of the invention, a method for treating or preventingCVD complications, such as AMI or CVD death, in a subject in needthereof is provided. The method comprises administering atherapeutically effective dose of a drug capable of modulating one ormore of the lipid concentration(s), lipid-lipid concentration ratio(s)or lipid-clinical concentration ratio(s) described in Tables 3-8,wherein the dose is such that said one or more lipid concentration(s),lipid-lipid concentration ratio(s) or lipid-clinical concentrationratio(s) in a sample of said subject does not significantly differ whencompared to (a) corresponding lipid concentration(s), (a) correspondinglipid-lipid concentration ratio(s) or (a) corresponding lipid-clinicalconcentration ratio(s) in a control, e.g., a control sample. In apreferred embodiment, the drug is a statin or another HMG-CoA reductaseinhibitor. Particularly preferred statins in this regard areatorvastatin, cerivastatin, fluvastatin, fluvastatin XL, lovastatin,pitavastatin, pravastatin, rosuvastatin or simvastatin. In anotherpreferred embodiment, the drug is niacin (nicotinic acid); a cholesterolabsorption inhibitor, such as ezetimibe or SCH-48461; a cholesterylester transfer protein (CETP) inhibitor, such as torcetrapib,anacetrapib or JTT-705; a bile acids sequestrant, such as colesevelam,cholestyramine or colestipol; or a fibrate, such as fenofibrate,gemfibrozil, clofibrate, or bezafibrate. Alternatively, it may also be aphytosterol or a PCSK9 inhibitor.

Also part of the present invention is a lipid as described herein, e.g.a lipid from any of Tables 3, 4, 5, 6, 7 or 8, for use in preventing ortreating a subject at risk to develop CVD complications such as AMI orCVD death. The said lipid may be taken as a dietary supplement or amedicament. A corresponding method of treatment is likewise encompassed.Likewise, the invention also encompasses a modulator for use formodulating a lipid concentration, lipid-lipid concentration ratio orlipid-clinical concentration ratio as described and/or claimed herein,e.g., in Tables 3-8, in a subject at risk to develop CVD and/or one ormore of its complications such as AMI or CVD death. A correspondingmethod of treatment is likewise encompassed. In a further embodiment,the said modulator is a small molecule, an antisense RNA, a smallinterfering RNA (siRNA) or a natural or modified lipid.

Alternatively, the modulator affects the activity, functionality orconcentration of an enzyme, wherein said enzyme catalyzes a reactionthat produces or degrades any one of the lipids in Tables 3-8.Similarly, the present invention relates to a preventing or treating asubject at risk to develop CVD complications such as AMI or CVD death,using, or administering a modulator, wherein the modulator affects theactivity, functionality or concentration of an enzyme, wherein saidenzyme catalyzes a reaction that produces or degrades any one of thelipids in Tables 3-8.

In one embodiment of the present invention, an antibody against any oneof the lipids in Tables 3-8 is used for predicting one or more CVDcomplications such as AMI or CVD death. In another embodiment of theinvention, the antibody may be used for preventing or treating one ormore of the above complications in a subject.

Any of the methods, drugs, lipids, modulators or antibodies of thepresent invention may be used for a subject which is at risk to developor has suffered from one or more CVD complications such as acutemyocardial infarction and/or is at risk of cardiovascular death. For thepurposes of the invention, CVD complication(s) includes severe CVDcomplication(s), particularly death.

Also encompassed by the present invention is a kit for predicting CVDand/or one or more of it complications, or for performing the methods oruses described and/or claimed herein, wherein the kit comprises reagentsand reference compounds. The reference compounds may be one or more ofthe following, but are not limited to: (a) (a) lipid standard(s) chosenfrom the lipids in Tables 3 to 8, (b) one or more control markers (forexample, a lipid or lipids, preferably a lipid corresponding to any ofthe lipidomic markers described and/or claimed herein, or (an)otherlipid(s), e.g., total PC, or another molecule, e.g., a protein; c)positive and/or negative controls; d) internal and/or externalstandards; e) calibration line controls; (f) an antibody or otherbinding moiety capable of binding any one of the lipids in Tables 3 to8. The reagents are solution(s), solvent(s), and/or buffer(s) useful forperforming said methods or uses.

In one embodiment of the invention, a kit is provided for predicting CVDand/or one or more of its complications, or for performing the methodsof the invention, wherein the kit comprises (a) (a) lipid standard(s)chosen from the lipids in Tables 3 to 8, and optionally one or morefurther reference compound(s) selected from: (b) one or more controlmarkers (for example, a lipid or lipids, preferably a lipidcorresponding to any of the lipidomic markers described and/or claimedherein, or another lipid(s), e.g., total PC, or another molecule, e.g.,a protein); c) positive and/or negative controls; d) internal and/orexternal standards, which may or may not be chemically modified, taggedor non-endogenously occurring molecules in human; e) calibration linecontrols; and (f) an agent, optionally an antibody, capable of bindingany one of the lipids in Tables 3 to 8, and (g) (a) reagent(s) forperforming said methods or uses.

Preferred kits according to the invention comprise, for example, thefollowing combinations of the above listed constituents: (a) and (b),and optionally (g); (a) and (c), and optionally (g); (a) and (d), andoptionally (g); (a) and (e), and optionally (g); (a) and (f), andoptionally (g); (a), (b) and (c), and optionally (g); (a), (c) and (d),and optionally (g); (a), (d) and (e), and optionally (g); or (a), (e)and (f), and optionally (g).

In one preferred embodiment, the one or more control marker(s) of theclaimed kit is/are (a) molecule(s) that is/are regularly measured in aclinical setting. For example, preferred are embodiments wherein the oneor more said control marker(s) is CK.

Preferably, the kit is used for predicting CVD complications, whereinthe lipid concentration(s), lipid ratio(s) or (a) lipid combination(s)thereof in a sample from a subject is (are) determined by using massspectrometry. The sample may be subjected to purification and/or othersample pre-preparation step(s) before mass spectrometry analysis. Thepurification step may be, but is not limited to chromatography, forexample, high performance liquid chromatography (HPLC) and/or ultra highperformance liquid chromatography (UHPLC). The sample pre-preparationstep may be, but is not limited to solid-phase extraction (SPE),derivatization, liquid-liquid extraction and/or lipoproteinfractionation. The said mass spectrometry determination may be done bytandem mass spectrometry.

As mentioned above, for the purposes of the present invention, a controlsample may be obtained from (a) CAD patient(s) or a group of CADpatients that has/have remained free of any major CVD complicationse.g., by mixing a variety of samples from said population. If a group ofCAD patients is used then several lipid profiles from a population arecombined and the lipidomic marker is created from this combination. Thelevels or amounts of the individual lipids or the lipid-lipidconcentration ratios or lipid-clinical concentration ratios in thesample from a subject are compared to the levels or amounts of thelipids or lipid ratios in the control for determining the risk of one ormore of CVD complications, such as AMI or CVD death, in said subject.

In one embodiment, the control is a sample from (a) CAD patient(s)wherein the control sample is from (a) CAD patient(s) or a group of CADpatients with no history of major CVD events and who is/are notundergoing statin treatment. The control CAD patient(s) not undergoingstatin treatment may or may not have a type 2 diabetes mellitus. It mayalso be a sample that represents a combination of samples from a CADpatient population not undergoing statin treatment with no history ofmajor CVD events. Alternatively, the control may be a set of dataconcerning a lipidomic marker in accordance with the present invention,e.g., information on the concentration of (a) lipid(s), lipid-lipidconcentration ratio(s), or lipid-clinical concentration ratio(s) inaccordance with the present invention in a sample when taken from (a)CAD patient(s) not undergoing statin treatment with no history of majorCVD events, or in a combination of samples taken from a CAD patientpopulation not undergoing statin treatment with no history of major CVDevents. Said information, and thus the corresponding set of data, mayhave been previously determined, calculated or extrapolated, or may haveyet to be determined, calculated or extrapolated, or may also be takenfrom the literature.

Alternatively, the control is a sample from (a) CAD patient(s) whereinthe control sample is from (a) CAD patient(s) or a group of CAD patientswith no history of major CVD events and who is/are undergoing statintreatment. The control CAD patient(s) undergoing statin treatment may ormay not have a type 2 diabetes mellitus. It may also be a sample thatrepresents a combination of samples from a CAD patient populationundergoing statin treatment with no history of major CVD events.Alternatively, the control may be a set of data concerning a lipidomicmarker in accordance with the present invention, e.g., information onthe concentration of (a) lipid(s), lipid-lipid concentration ratio(s),or lipid-clinical concentration ratio(s) in accordance with the presentinvention in a sample when taken from (a) CAD patient(s) undergoingstatin treatment with no history of major CVD events, or in acombination of samples taken from a CAD patient population undergoingstatin treatment with no history of major CVD events. Said information,and thus the corresponding set of data, may have been previouslydetermined, calculated or extrapolated, or may have yet to bedetermined, calculated or extrapolated, or may also be taken from theliterature.

Preferably, the control sample is blood, plasma, serum, urine or tissue,or a lipoprotein fraction thereof.

In another aspect, the present invention relates to a statin or a lipidlowering drug for use in the treatment of a subject at risk to developone or more CVD complications, such as acute myocardial infarction (AMI)and/or CVD death, wherein said subject would be identified as being atrisk to develop one or more CVD complications when applying any of themethods, drugs, lipids, modulators, kits or uses described and/orclaimed herein. Similarly, the present invention relates to a method oftreating a subject at risk to develop one or more CVD complications witha statin or a lipid lowering drug, wherein said subject would beidentified as being at risk to develop one or more CVD complicationswhen applying any of the methods, drugs, lipids, modulators, kits oruses described and/or claimed herein.

In a further embodiment, the present invention relates to a statin or alipid lowering drug for use in the treatment of a subject at risk todevelop one or more CVD complications, such as AMI and/or CVD death,wherein said subject actually has been identified as being at risk todevelop one or more CVD complications by any of the methods, drugs,lipids, modulators, kits or uses described and/or claimed herein.Similarly, the present invention relates to a method of treating asubject at risk to develop one or more CVD complications with a statinor a lipid lowering drug, wherein said subject actually has beenidentified as being at risk to develop one or more CVD complications byany of the methods, drugs, lipids, modulators, kits or uses describedand/or claimed herein.

In yet another aspect, the present invention relates to a statin or alipid lowering drug for use in the treatment of a subject at risk todevelop one or more CVD complications, such as AMI and/or CVD death,wherein said subject would be identified as not being at risk to developor as not suffering from one or more CVD complications when applying anyof the methods, drugs, lipids, modulators, kits or uses described and/orclaimed herein. Similarly, the present invention relates to a method oftreating a subject at risk to develop one or more CVD complications witha statin or a lipid lowering drug, wherein said subject would beidentified as not being at risk to develop one or more CVD complicationswhen applying any of the methods, drugs, lipids, modulators, kits oruses described and/or claimed herein.

In a further embodiment, the present invention relates to a statin or alipid lowering drug for use in the treatment of a subject at risk todevelop one or more CVD complications, such as AMI and/or CVD death,wherein said subject actually has been identified as not being at riskto develop or as not suffering from statin-induced muscle toxicity byany of the methods, drugs, lipids, modulators, kits or uses describedand/or claimed herein. Similarly, the present invention relates to amethod of treating a subject at risk to develop one or more CVDcomplications with a statin or a lipid lowering drug, wherein saidsubject actually has been identified as not being at risk to develop oneor more CVD complications by any of the methods, drugs, lipids,modulators, kits or uses described and/or claimed herein.

In the present invention herein, lipid biomarker concentrations havebeen measured and quantified in patients with documented CAD who did notshow fatal outcomes during the follow-up period (3 years) and inhigh-risk CAD patients who died due to cardiovascular events during thefollow-up period. This invention thus enables accurate usage of thelipid-based biomarkers to identify high risk CVD/CAD patients. Anotherlayer of accuracy was reached through a careful patient selection sinceit is important to control for factors which may affect the lipidconcentration read-outs. Unlike the previous efforts described above, weused specific targeted platforms on a singular technology set-up toanalyze lipid species in particular.

The technology and the way it was applied in the context of theinventive teaching presented herein is set apart from similar efforts inthe field inter alia due to the following criteria. In samplepreparation, samples are strictly controlled and treated identically toavoid potential artifacts that could arise from improper handling. Inconnection with the present invention, samples were carefully thawedslowly on ice and directly thereafter subjected to a custom-madeautomated lipid extraction which possesses currently the highestprecision in liquid handling, therefore minimizing potential errors.Furthermore, sample freeze-thaw cycles were strictly controlled sincethis can dramatically affect the lipid stabilities. The automated lipidextraction is based on the method by Folch and colleagues (Folch J, etal: A simple method for the isolation and purification of total lipidsfrom animal tissues. J Biol Chem 1957, 226(1):497-509) which useschloroform and methanol. This method is preferred when a wide range,from polar to non-polar, of lipid classes are to be extracted withoptimal recoveries thus preventing the loss of lipid species. Lipidclass specific non-endogenous lipids, when applicable, were used asinternal standards to gain highest precision in identification(minimizing false positives) and quantification of monitored molecularlipid species. In this way absolute or semi-absolute amounts ofendogenous molecular lipids were determined with the highest precisionthat can be achieved with today's technologies. The endogenous lipidsand respective standards were monitored at the molecular lipid level. Inthis way, not only false positive identifications were minimized, butmolecular lipids could be precisely determined and quantified. Analysisquality was strictly controlled using a novel quality control system.This was mainly controlled by multiple internal standards (IS), externalstandards (ES), IS/ES ratios, and instrument control samples. Bystringently controlling these components, technical and biologicaloutliers were readily identified and rejected from further analysis. Toobtain best precision in sensitivity, selectivity and quantification foreach molecular lipid different targeted platforms were used. Some lipidsare best analyzed using high performance liquid chromatography (HPLC) orultra high performance liquid chromatography (UHPLC) combined with massspectrometry based multiple reaction monitoring (MRM) whereas others arebest analyzed by direct infusion in combination with massspectrometry-based precursor ion scanning and neutral loss scanningtechniques.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Coronary vascular disease/cardiovascular disease (CVD) has its generalmeaning in the art and is used to classify numerous conditions thataffect the heart, heart valves, blood, and vasculature of the body,including CAD. In the present invention the terms CVD and CAD may beused interchangeably. For the purposes of the invention, CVD/CADpatients, in one embodiment, exclude patients with Acute CoronarySyndrome (ACS). In an alternative embodiment, ACS is included inCVD/CAD. Cardiovascular diseases in accordance with the presentinvention include endothelial dysfunction, coronary artery disease,angina pectoris, myocardial infarction, atherosclerosis, congestiveheart failure, hypertension, cerebrovascular disease, stroke, transientischemic attacks, deep vein thrombosis, peripheral artery disease,cardiomyopathy, arrhythmias, aortic stenosis, and aneurysm. Suchdiseases frequently involve atherosclerosis. In a preferred embodimentof the invention, the cardiovascular disease is a cardiovascular diseaseassociated with atherosclerosis.

CAD is coronary artery disease, AMI is acute myocardial infarction, ACSis acute coronary syndrome, CAC is coronary artery calcification, RCT isreverse cholesterol transport, LDL is low density lipoprotein, HDL ishigh density lipoprotein, LDL-C is low density lipoprotein cholesterol,HDL-C is high density lipoprotein cholesterol, ApoA is Apolipoprotein A,ApoB is Apolipoprotein B, ApoC is apolipoprotein C, MS is massspectrometry, HPLC is high performance liquid chromatography, UHPLC isultra high performance liquid chromatography and UPLC is ultraperformance liquid chromatography.

As used herein, “a subject” includes all mammals, including withoutlimitation humans, but also non-human primates, dogs, cats, horses,sheep, goats, cows, rabbits, pigs and rodents. A particularly preferred“subject” is a human.

A “sample” is defined as any biological sample obtained from a subjector a group or population of subjects. For the purposes of the presentinvention, the biological sample may be whole blood, blood serum, bloodplasma or a fraction of blood; e.g., a lipoprotein fraction. It may alsobe a tissue sample. However, a preferred embodiment is wherein thebiological sample is plasma or serum. Taking a blood sample of a patientis part of normal clinical practice. The blood sample can be taken inconnection with e.g. measuring the cholesterol levels in the patients.The collected blood sample can be prepared and serum or plasma can beseparated with techniques well known to a person skilled in the art.Vena blood samples can be collected from patients using a needle and aBD Vacutainer® Plastic Tubes or Vacutainer® Plus Plastic Tubes (BDVacutainer® SST™ Tubes contain spray-coated silica and a polymer gel forserum separation). Serum can be separated by centrifugation at 1300 RCFfor 10 min at room temperature and stored in small plastic tubes at −80°C. The lipoprotein fractions may be separated by precipitation,ultracentrifugation, by chromatography or by gel filtration with methodswell known in the art.

For the purposes of the present invention, lipids from the Lipidomicanalysis were named according to the following nomenclature: CE ischolesteryl ester, Cer is ceramide, DAG is diacylglycerol, PC O isether-linked PC, Gb3 is globotriaosylceramide, GlcCer is galactosyl- andglucosylceramides, LacCer is lactosylceramides, LPC islysophosphatidylcholine, PC is Phosphatidylcholine, PE isPhosphatidylethanolamine, PI is Phosphatidylinositol and SM isSphingomyelin.

The nomenclature X:Y indicates, X number of total carbon atoms in thefatty acid(s) portions of the molecule, and Y the total number of doublebonds in the fatty acid portion(s) of the molecule.

The nomenclature A/B indicates, for a molecule of DAG and PC, A and Btypes of fatty acid moieties attached to the glycerol backbone of themolecule.

The nomenclature (dC/A) indicates, for a molecule of Cer, Gb, GlcCer,LacCer and SM, C the type of long-chain base with an amide-linked, A,fatty acid moiety.

According to the invention, the treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject not undergoingstatin treatment, does not involve treatment with a statin. For example,the said treatment may be one with a niacin (nicotinic acid); acholesterol absorption inhibitor, such as ezetimibe or SCH-48461; acholesteryl ester transfer protein (CETP) inhibitor, such astorcetrapib, anacetrapib or JTT-705; a bile acids sequestrant such ascolesevelam, cholestyramine and colestipol; a fibrate, such asfenofibrate, gemfibrozil, clofibrate, and bezafibrate; a phytosterol, ora PCSK9 inhibitor.

The wording “compared to a control sample” as used herein will beunderstood to include embodiments where control samples are actuallyanalyzed in respect of a lipidomic marker of interest, i.e., in respectof the concentration of one or more of the lipid(s), the lipid-lipidconcentration ratios, or the lipid-clinical concentration ratios orcombinations thereof as specifically described and/or claimed herein inconnection with the various aspects and embodiments of the presentinvention. It will be appreciated, however, that the above wording alsoincludes embodiments where the corresponding information on saidlipidomic marker in said control sample is merely taken from theliterature, or has been previously determined, calculated orextrapolated, or is yet to be determined, calculated or extrapolated.

As used herein, the term “antibody” includes monoclonal and polyclonalantibodies, whole antibodies, antibody fragments, and antibodysub-fragments that exhibit specific binding to a said lipid. Thus,suitable “antibodies” can be whole immunoglobulins of any class, e.g.,IgG, IgM, IgA, IgD, IgE, chimeric antibodies or hybrid antibodies withdual or multiple antigen or epitope specificities, or fragments, e.g.,F(ab′)₂, Fab′, Fab and the like, including hybrid fragments, andadditionally includes any immunoglobulin or any natural, synthetic orgenetically engineered protein that acts like an antibody by binding toa specific antigen to form a complex. The term “antibody” encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab fragments, F(ab′)₂, an Fd fragment, an Fv fragment, a dAb (single(variable) domain antibody), or a nanobody) as well as completeantibodies. For example, Fab molecules can be expressed and assembled ina genetically transformed host like E. coli. A lambda vector system isavailable thus to express a population of Fab's with a potentialdiversity equal to or exceeding that of the predecessor antibody. SeeHuse W D, et al., Science 1989, 246:1275-81. Such Fab's are included inthe definition of “antibody.” The ability of a given molecule, includingan antibody fragment or sub-fragment, to act like an antibody andspecifically bind to a specific antigen can be determined by bindingassays known in the art, for example, using the antigen of interest asthe binding partner.

Antibodies against lipids in accordance with the present invention maybe prepared by methods well known to those skilled in the art. Forexample, mice may be immunized with a lipid with adjuvant. Splenocytesare harvested as a pool from the mice that were administered 3immunizations at 2-week intervals with test bleeds performed onalternate weeks for serum antibody titers. Splenocytes are prepared as 3aliquots that are either used immediately in fusion experiments orstored in liquid nitrogen for use in future fusions.

Fusion experiments are then performed according to the procedure ofStewart & Fuller, J. Immunol. Methods 1989, 123:45-53. Supernatants fromwells with growing hybrids are screened by enzyme-linked immunosorbentassay (ELISA) for monoclonal antibody (MAb) secretors on 96-well ELISAplates coated with the said lipid. ELISA positive cultures are cloned bylimiting dilutions, typically resulting in hybridomas established fromsingle colonies after 2 serial cloning experiments.

EXAMPLES Example 1 Materials and Methods

This study is a sub-cohort of the LURIC study that is a large scaleprospective study on cardiovascular epidemiology. LURIC databasecontains clinical information over 3000 patients including baselinecoronary angiography, clinically used biomarker data and also e.g. CVDmortality data for the follow-up period (3 years). In this biomarkerstudy the inventors compared CAD cases not undergoing statin treatment(n=123) that died during the follow-up due to CVD with patients (n=96)having a stable CAD also not undergoing statin treatment. Subjects witha significant atherosclerosis level in the angiogram but no CVD relateddeath during the follow-up were used as controls, while the case grouphad similarly a significant atherosclerosis based on the angiography atbaseline and in addition they died during the follow-up due to acutecardiovascular events. A statistical analysis was performed separatelyfor cases (n=55) and controls (n=46) without diabetes and for cases(n=68) and controls (n=50) having diabetes. The clinical characteristicsfor these two groups are described in Tables 1 and 2.

TABLE 1 Background characteristics of subjects not undergoing statintreatment and not having type 2 diabetes mellitus Clinicalcharacteristics Controls (n = 46) Cases (n = 55) DM2 patients 0 0Hypertensive patients 27 (58.7%) 32 (58.2%) Smokers (active or quit lessthan 3 years 13 (28.3%) 17 (30.9%) before sampling) Statin users 0 0 Age66.9 67.6 Apolipoprotein A-I 134.0 125.2 Apolipoprotein B 104.0 106.8BMI 27.2 26.3 HDL cholesterol 41.3 38.2 LDL cholesterol 121.8 124.8Lipoprotein(a) 22.8 26.4 Supersensitive C-reactive protein 7.4 17.2Total cholesterol 194.8 195.5 Triglycerides 138.0 145.6

TABLE 2 Background characteristics of subjects not undergoing statintreatment and having type 2 diabetes mellitus Clinical characteristicsControls (n = 50) Cases (n = 68) DM2 patients  50 (100%) 68 (100%) Hypertensive patients 35 (70%) 41 (60.4%) Smokers (active or quit lessthan 18 (36%) 21 (30.9%) 3 years before sampling) Statin users 0 0 Age65.1 69.0 apolipoprotein A-I 124.0 116.9 apolipoprotein B 115.5 106.7bmi 27.8 28.0 HDL cholesterol 35.2 34.1 LDL cholesterol 126.0 114.4lipoprotein(a) 32.0 15.6 supersensitive C-reactive protein 5.3 11.5total cholesterol 205.6 189.9 triglycerides 194.4 183.3 Definition ofCases: All cases had significant artery disease (>=20% stenosis) incoronary angiogram and they all died due to CVD during the follow-up.Thus, these CAD patients have an elevated risk for CVD outcomes.Majority (75%) of cases, were non-ACS patients. Definition of Controls:All controls had significant artery disease (>=20% stenosis) in coronaryangiogram, but they did not die due to CVD during the follow-up. Thecontrol subjects did not have any history of MI or stroke before thebaseline evaluation. Thus, these patients can be considered to be CADpatients with a low risk for CVD outcomes.

Example 2 Analytical Methods

Mass Spectrometry Driven Lipidomics

Direct infusion coupled to tandem mass spectrometry, i.e. shotgunlipidomics, and two liquid chromatography tandem mass spectrometry(LC-MS/MS) approaches, i.e. ceramide and cerebroside lipidomics andganglioside lipidomics, were used to identify lipid biomarkers forcoronary artery disease (CVD) risk by analyzing molecular lipid speciesin human serum, plasma, and carotid artery plaques. The applied methodswere optimized especially for quantification of molecular cholesterylesters (CE), phosphatidylcholines (PC), lysophosphatidylcholines (LPC)and other lysophospholipids (LPL), ether-linked phosphatidylcholines (PCO) and other ether-linked phospholipids (PL O), phosphatidylserines(PS), phosphatidylethanolamines (PE), phosphatidylglycerols (PG),phosphatidylinositols (PI), phosphatidic acids (PA), diacylglycerols(DAG), ceramides (Cer), glucosylceramides (GlcCer),Globotriaosylceramide (Gb3) and lactosylceramides (LacCer).

The following materials were used according to the methods. HPLC orLC-MS grade of chloroform, methanol, water, acetonitrile, formic acid,methanol, isopropanol, ammonium acetate, acetic acid, potassium chlorideand butylated hydroxytoluene (BHT) were purchased from Sigma-Aldrich(St. Louis, Mo., USA).

HPLC column (Acquity BEH C18, 2.1×50 mm id 1.7 μm) was purchased fromWaters (Milford, Mass., USA). HPLC pre-column (Widepore C18 4×2.0 mm)was purchased from Phenomenex (Torrance, Calif., USA). All labware usedfor the extraction were resistant to chloroform. Aerosol resistantfilter tips (Molecular BioProducts) and Eppendorf 2 ml safe-lock tubes,96-well twin.tec PCR plates, and Pierce-it-lite thermo-sealing foilswere purchased from VWR International (West Chester, Pa., USA). CO-REFilter Tips and 96-well 2 ml Whatman Uniplates were purchased fromHamilton Robotics (Bonaduz, Switzerland). Synthetic lipid standards werepurchased from Avanti Polar Lipids (Alabaster, Ala., USA) and fromMatreya (Pleasant Gap, Pa., USA).

Lipids were extracted in chloroform:methanol according to the followingprotocols. Samples were spiked with known amounts of non-endogenoussynthetic internal standards for data normalization and endogenous lipidquantification. Post-extract spiked non-endogenous synthetic externalstandards were used for quality controlling. Stock solutions ofstandards were prepared by dissolving appropriately weighed amounts ofeach standard in chloroform:methanol (2:1, V:V) to achieve a finalconcentration of 500 μM. An internal standard mixture containing each ofthe standard stock was created and used in lipid extraction.

Samples and quality control samples for each extraction batch werethawed on ice. The carotid artery plaque samples were weighed on ice byusing a cryo-box and homogenized in ice-cold 70% methanol in water. TheMixer Mill 301 Teflon adapters were kept at −20° C. Homogenization wasperformed at 15-25 Hz for 2-15 minutes with Mixer Mill 301 (Retch GmbH,Germany).

Lipid extraction of human samples was carried out in automated fashionusing a Hamilton MICROLAB STAR system (Hamilton Robotics, Switzerland).Well-mixed samples were aliquoted into a 96-well 2 ml Whatman Uniplatecontaining ice-cold methanol and 0.1% BHT. 5 μl of serum and plasma and30 μl of carotid artery plaques were used for shotgun- and ceramide andcerebroside lipidomics and 100 μl of serum and plasma and 200 μl ofcarotid artery plaques was used for ganglioside lipidomics. The sampleswere mixed thoroughly after each step in the extraction protocol. Theextraction proceeded at room temperature by adding an appropriate volumeof internal standard mixture and chloroform, and methanol and water inthe case of ganglioside lipidomics. In shotgun and ceramide andcerebroside lipidomics, the organic phase separation was facilitated byadding 20 mM acetic acid and centrifuging the plate for 5 min at 500×g.The organic phase was transferred into a new 96-well 2 ml WhatmanUniplate. The remaining water-containing phase was washed by addingappropriate volume of chloroform followed by centrifugation. The twoorganic phases were pooled and evaporated under N₂ until dryness. Thelipid extracts were then re-dissolved in chloroform:methanol (1:2, v:v)including the addition of the synthetic external standard.

In shotgun lipidomics, lipid extracts were analyzed on a hybrid triplequadrupole/linear ion trap mass spectrometer (QTRAP 5500, AB Sciex)equipped with a robotic nanoflow ion source (NanoMate HD, AdvionBiosciences). The instruments were operated in positive and negative ionmodes. In positive ion the spray voltage was set to 1.0 to 1.4 kV and innegative ion mode to −1.0 to −1.4 kV. A gas pressure of 0.3-0.8 psi wasused and the interface heater was set at 60° C. The collision energy(CE) and declustering potential (DP) was optimized for each lipid classusing synthetic standards. The mass spectrometer was operated in unitresolution mode using a scan speed of 200 Da/s. Molecular lipids wereanalyzed in both positive and negative ion modes using multipleprecursor ion scanning (MPIS) and neutral loss scanning (NLS) asdescribed by Ståhlman and colleagues (Ståhlmna M, et al. High-throughputshotgun lipidomics by quadrupole time-of-flight mass spectrometry. JChromatogr B Analyt Technol Biomed Life Sci 2009).

In ceramide and cerebroside lipidomics, the high performance liquidchromatography (HPLC) analyses were conducted in the following way.Chromatographic apparatus consisted of a CTC HTC PAL autosampler (CTCAnalytics AG, Switzerland), a Rheos Allegro UHPLC pump (Flux InstrumentsAG, Switzerland), an external column heater set to 60° C. for ceramideand cerebroside lipidomics and 45° C. for ganglioside lipidomics, andthe Acquity BEH C18 column with an in-line pre-column. The extractedsamples, 10 μl of each, were injected into the pre-column followed bythe analytical column and delivered to the mass spectrometer at a flowrate of 500 μl/min. In ceramide and cerebroside lipidomics, A gradientwas used for lipid analyte separation with solvent A comprising 10 mMammonium acetate in HPLC grade water containing 0.1% formic acid andsolvent B of 10 mM ammonium acetate in acetonitrile:isopropanol (4:3,V:V) containing 0.1% formic acid. The gradient was constructed in thefollowing way: 0 min—65% B; 2 min—65% B; 2.5 min—75% B; 17.5 min—100% B;22.5 min—100% B; 22.6 min—65% B; 25 min—65% B.

The lipid extracts were analyzed by HPLC-MS/MS. The MS analysis wasperformed on a hybrid triple quadrupole/linear ion trap massspectrometer equipped with the Turbo V™ Ion Source (4000 QTRAP, ABSciex). The instrument was operating in positive and negative ion modes.The ion source voltage was set to 5500V for ceramide and cerebrosidelipidomics and to −4500V for ganglioside lipidomics, and sourcetemperature at 400° C. The collision energy (CE) and declusteringpotential (DP) was optimized for each lipid class using syntheticstandards. A 20 sec dwell time was applied for each scan. Multiplereaction monitoring (MRM) scan mode was applied and based on thedescription by Sullards and colleagues (Sullards M C, et al:Structure-specific, quantitative methods for analysis of sphingolipidsby liquid chromatography-tandem mass spectrometry: “inside-out”sphingolipidomics. Methods Enzymol 2007).

The data processing was done in the following way. Initially theretention time (in LC mode) and identification of each peak was doneusing endogenous standards and by Information Dependent Acquisition(IDA) experiments where applicable. The raw data were processedaccording to peak detected and retention time (in LC mode) in automatedfashion. A stringent cutoff was applied for separating background noisefrom actual lipid peaks. Each sample was controlled and only acceptedwhen fulfilling the stringent acceptance criteria. Peak area counts(cps) of detected peaks were converted into a list of correspondinglipid names. Lipids were normalized to their respective internalstandard and sample volume or tissue weight to retrieve theirconcentrations.

Several quality controls were used in the lipidomic analyses. Acalibration line using synthetic or isolated standards was obtainedprior to sample analysis. Synthetic standards were chosen based onapplication and had similar properties to the endogenous lipids oranalyte(s) of interest. The calibration line consisted of a minimum offive standards points covering the expected quantification range. Asample extracted without standard and standards extracted with nomatrix, were included with the calibration line.

The calibration line was used to determine the dynamic quantificationrange for each lipid class monitored, e.g., the linear quantificationlimits. As the internal standards used behave in the same way asendogenous lipids they were used for quantifying endogenous lipidspecies. The calibration lines were based on the same internal standardsthat were used for quantification of the endogenous lipids.

In each sample extracted for lipids, the ratio of synthetic internalstandards (IS) to corresponding post-extract spiked external standard(ES) was determined. The peak area (cps) ratio of internal to externalstandard (IS/ES) was used for calculating the Coefficient of Variation(CV) across all samples. The IS/ES ratio enabled the calculation oflipid extraction recovery.

Instrument control (IC) was included at the start, middle and end ofeach run. IC sample analyzed was an extracted reference plasma sampleand a set of standards to monitor the instrument's performance, i.e.,the intra- and inter-assay variation.

For each platform, a stringent cutoff was applied for separatingbackground noise from actual lipid peaks. Each sample was controlled andonly accepted when fulfilling the stringent acceptance criteria. Massesand counts of detected peaks were converted into a list of correspondinglipid names. Lipids were normalized to their respective internalstandard and sample volume to retrieve their concentrations.

Statistical Analyses

Percentage changes in lipid concentrations between control and casegroups were calculated as follows:100*(AVG[C] in case group−AVG[C] in control group)/AVG[C] in controlgroup

Statistical significance was assigned based on standard t-test p-values.

In addition, ROC curves were used for finding lipid molecules andconcentration cutoffs that separate the best cases from controls.Selectivity is calculated as a number of correctly identified casesdivided by the total number of cases. Specificity is calculated as anumber of correctly identified controls divided by the total number ofcontrols. Selectivity and specificity was calculated for each lipidconcentration, lipid to lipid ratio and ratio of lipid to clinicalconcentrations.

Example 3 Ethics

The LURIC study was approved by the ethics review committee at the“Landesärztekammer Rheinland-Pfalz” (Mainz, Germany). Written informedconsent was obtained from each of the participants.

Results

In this LURIC study sub-cohort, the traditional biomarkers includingLDL-cholesterol and HDL-cholesterol concentrations were practicallyidentical in both groups and therefore were not predictive ofCVD-related for predicting severe CVD/CAD-associated complications,including AMI, stroke and CVD death in this study.

Multiple lipidomic markers appeared as significant predictors of severeCVD/CAD-associated complications (Tables 3-8). A total of 162 molecularlipids were quantified. The significant predictors were selected basedon the top fifty candidates from each category, when available. Thebiomarker candidates were selected according to the following criteria:t-test p-value≦0.05 or sensitivity≧60% and specificity≧60%. Fromtraditional clinical chemistry only apolipoprotein A1 and totalcholesterol reached statistical significance with p-value lower than0.05, but % change was less than 10% between controls and cases, otherclinical values did not show any statistical significance. Thepredictive value of new lipidomic biomarkers was increased when theirlevels were expressed as distinct lipid-lipid concentration ratios orlipid-clinical ratios (e.g. LDL-C or HDL-C).

Furthermore, to demonstrate improved diagnostic potential, logisticmodels were fitted in order to find different combinations of lipidsthat could separate cases and controls from each other. The lipids wereset as possible explanatory variables and model was selected usingstepwise method with different entry and stay significance levels. Themarkers with best diagnostic potential are listed in Table 8.

Importance of Detailed Molecular Lipid Analyses

Recent evolvement of mass spectrometry driven lipid analysis approacheshas made it possible to resolve complex lipidomes to their molecularlipid species level at high-throughput and quality required for analysesof clinical cohorts. As a result of the high sensitivity and selectivityof the methods, a lipidome-wide analysis of minute sample amounts hasbecome feasible. Present technologies are capable of identifying lipidswith different sum compositions, i.e. phosphatidylcholine (PC) 34:1, butmore important is the identification of molecular lipid species, e.g. PC16:0/18:1. In the latter analysis, information of the type of fattyacids and their positions attached to the glycerol backbone making upthe particular PC molecule is retrieved.

The seminal work of Shinzawa-Itoh and colleagues showed by highlysophisticated experiments that the oxygen transfer mechanism incytochrome c oxidase requires a specific phosphatidylglycerol molecularlipid with palmitate and vaccenate at the sn-1 and sn-2 positionsrespectively on the glycerol backbone (Shinzawa-Itoh K, Aoyama H,Muramoto K et al: Structures and physiological roles of 13 integrallipids of bovine heart cytochrome c oxidase. EMBO J 2007, 26:1713-1725).In line with other studies, this undoubtedly indicates that the lipidstructure is an essential determinant of the biological effect.Therefore, molecular lipidomics is an essential for biomarker discovery.Figure 1 illustrates the importance of molecular lipid data by comparingthe biomarker value of two PC and LacCer molecules in predicting CVDmortality in the LURIC cohort. The data reveals that whileLacCer(d18:1/20:0) is a significant CVD predictor, LacCer (di8:1/18:16:0) has low biomarker potential. In addition, two PC moleculesPC (18:0/20:4) and PC (18:0/16:0) have even opposite effects on CVDcomplications. Thus, it is always necessary to identify and quantify alllipid species for lipid classes of interest including but not limited tocholesterol esters, different phospholipid classes, ceramides,cerebrosides (lactosylceramides, glycosylceramides,globotriaosylceramides).

TABLE 3 Significant markers (lipid-lipid concentration ratios and lipid-clinical concentration ratios) for CVD patients not undergoing statintreatment. Lipid names, p-values, % change, AUC, Sensitivity andSpecificity are presented. Percentage Measurement name P-value changeAUC Sensitivity Specificity Lipid-lipid concentration ratios IncreasedGb3(d18:1/16:0)/PC 18:0/22:6 0.00054 50.7 0.73 69.0 66.7Cer(d18:1/18:0)/Cer(d18:1/24:0) 0.00208 43.6 0.73 71.7 71.7Cer(d18:1/20:0)/Cer(d18:1/24:0) 0.00056 35.0 0.70 68.6 60.9 SM(d18:1/16:0) (d18:1/15:1-OH)/SM (d18:1/24:0) 21.2 0.74 72.2 62.2(d18:1/23:1-OH) Cer(d18:1/22:0)/Cer(d18:1/24:0) 0.00153 12.1 0.70 74.565.2 Decreased Cer(d18:1/24:0)/PC 16:0/18:2 0.02011 −13.2 SM(d18:1/24:0) (d18:1/23:1-OH)/SM (d18:1/24:1) 0.00811 −15.1(d18:1/23:2-OH) Cer(d18:1/24:0)/Cer(d18:1/24:1) 0.00062 −16.9 0.70 74.560.9 Cer(d18:1/24:0)/SM (d18:1/14:0) (d18:1/13:1-OH) 0.01940 −17.3Cer(d18:1/24:0)/Gb3(d18:1/16:0) 0.00517 −20.1 0.68 64.8 65.2Cer(d18:1/24:0)/SM (d18:1/16:1) (d18:1/15:2-OH) 0.00327 −20.8 0.66 72.760.0 Lipid-clinical concentration ratios IncreasedGlcCer(d18:1/16:0)/total cholesterol 0.02241 21.6Gb3(d18:1/16:0)/apolipoprotein A-I 0.01039 21.2 0.63 64.8 60.9Gb3(d18:1/18:0)/apolipoprotein A-I 0.03763 20.3 Gb3(d18:1/16:0)/HDLcholesterol 0.04197 19.7 0.65 61.1 63.0 PC 16:0/18:2/total cholesterol1.6 0.55 63.6 60.9 Decreased Cer(d18:1/24:0)/supersensitive C-reactiveprotein −27.5 0.66 63.6 64.4 PC 18:0/22:6/supersensitive C-reactiveprotein −28.8 0.65 62.8 65.8

Table 4. Significant markers for CVD patients not undergoing statintreatment and not suffering from diabetes. Lipid names, p-values, %change, AUC, Sensitivity and Specificity values are presented. Table 4ashows significant lipid markers, Table 4b shows lipid-lipidconcentration ratio markers and Table 4c shows lipid-clinicalconcentration ratio markers.

4a) Significant lipid markers for CVD patients not undergoing statintreatment and not suffering from diabetes. Percentage Measurement nameP-value change AUC Sensitivity Specificity Increased CE 19:1 (oxCE682.6) 27.7 0.65 70.5 66.7 GlcCer(d18:1/16:0) 0.02176 22.7 SM(d18:1/18:1) 14.2 0.59 63.0 60.0 Decreased PC 18:0/22:6 0.00685 −20.30.67 74.4 61.5 SM (d18:1/23:1) (d18:1/22:2-OH) 0.01808 −22.1 0.64 60.061.0

4b) Significant lipid-lipid concentration ratio markers for CVD patientsnot undergoing statin treatment and not suffering from diabetes.Percentage Measurement name P-value change AUC Sensitivity SpecificityIncreased Cer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/22:2-OH) 0.00331 81.90.70 68.8 63.4 Cer(d18:1/16:0)/PC 18:0/22:6 0.00045 78.7 0.74 76.7 61.5Cer(d18:1/18:0)/PC 16:0/22:6 0.00259 74.5 0.71 67.3 67.4GlcCer(d18:1/16:0)/PC 18:0/22:6 0.00101 69.7 0.73 74.4 61.5GlcCer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/22:2-OH) 0.00462 68.5 0.7576.1 65.9 Cer(d18:1/16:0)/PC 16:0/22:6 0.00024 62.1 0.72 75.9 60.9Cer(d18:1/20:0)/SM (d18:1/23:1) (d18:1/22:2-OH) 0.00573 62.0 0.71 67.461.0 GlcCer(d18:1/18:0)/PC 16:0/22:6 0.00006 60.4 0.76 80.0 60.9 SM(d18:1/18:1)/SM (d18:1/23:1) (d18:1/22:2-OH) 60.2 0.76 81.6 63.4Cer(d18:1/20:0)/PC 16:0/22:6 0.00017 59.5 0.73 74.0 63.0 SM (d18:1/16:0)(d18:1/15:1-OH)/SM (d18:1/23:1) 57.1 0.73 74.0 63.4 (d18:1/22:2-OH) SM(d18:1/15:0) (d18:1/14:1-OH)/SM (d18:1/23:1) 55.1 0.73 78.0 63.4(d18:1/22:2-OH) GlcCer(d18:1/20:0)/SM (d18:1/23:1) (d18:1/22:2-OH) 54.90.71 78.3 61.0 Cer(d18:1/24:1)/PC 18:0/22:6 53.8 0.70 72.1 69.2 SM(d18:1/16:1) (d18:1/15:2-OH)/SM (d18:1/23:1) 52.9 0.75 82.0 61.0(d18:1/22:2-OH) GlcCer(d18:1/16:0)/PC 16:0/22:6 0.00081 52.6 0.72 75.960.9 LacCer(d18:1/22:0)/PC 16:0/22:6 0.00047 52.4 0.73 68.0 60.9LacCer(d18:1/22:0)/SM (d18:1/23:1) (d18:1/22:2-OH) 49.3 0.71 71.7 61.0GlcCer(d18:1/20:0)/PC 16:0/22:6 0.00042 48.1 0.74 76.0 60.9 CE 16:0/PC18:0/22:6 0.00008 47.2 0.76 81.4 64.1 Cer(d18:1/22:0)/PC 18:0/22:6 44.30.71 74.4 61.5 CE 18:2/PC 18:0/22:6 0.00025 43.5 0.75 67.4 64.1 CE18:1/PC 18:0/22:6 0.00106 42.6 0.73 79.1 61.5Cer(d18:1/16:0)/Cer(d18:1/24:0) 0.00011 42.0 0.75 81.8 60.9Gb3(d18:1/16:0)/PC 16:0/22:6 0.00039 39.4 0.70 71.7 60.9 CE 20:4/PC18:0/22:6 0.00053 38.1 0.70 62.8 61.5 CE 22:6/PC 18:0/22:6 0.00047 37.20.73 73.8 65.8 PC 16:0/16:0/PC 16:0/22:6 0.00058 35.4 CE 16:0/PC16:0/22:6 0.00023 33.8 0.71 68.5 63.0 CE 18:2/PC 16:0/22:6 0.00057 33.7Gb3(d18:1/24:0)/PC 16:0/22:6 0.00149 33.5 0.71 73.5 60.0 CE 18:1/PC16:0/22:6 0.00081 31.5 0.70 72.2 63.0 CE 20:4/PC 16:0/22:6 0.00059 30.7SM (d18:1/16:1) (d18:1/15:2-OH)/SM (d18:1/24:0) 26.2 0.72 74.1 62.2(d18:1/23:1-OH) Cer(d18:1/16:0)/Cer(d18:1/22:0) 0.00048 26.0 0.71 67.365.2 CE 16:0/Cer(d18:1/24:0) 0.00089 22.9 SM (d18:1/18:1)/SM(d18:1/24:0) (d18:1/23:1-OH) 21.5 0.72 77.4 60.0 GlcCer(d18:1/18:0)/SM(d18:1/24:0) (d18:1/23:1-OH) 21.3 0.72 70.0 62.2 SM (d18:1/15:0)(d18:1/14:1-OH)/SM (d18:1/24:0) 17.9 0.71 73.6 62.2 (d18:1/23:1-OH)Decreased Cer(d18:1/24:0)/GlcCer(d18:1/18:0) 0.00035 −22.3 0.70 60.865.2 Cer(d18:1/24:0)/GlcCer(d18:1/16:0) 0.00024 −23.8 0.70 67.3 60.9 PC16:0/22:6/SM (d18:1/16:0) (d18:1/15:1-OH) 0.00123 −24.4 0.72 75.9 60.0PC 18:0/22:6/SM (d18:1/14:0) (d18:1/13:1-OH) 0.00307 −26.4 0.71 74.460.5 PC 16:0/22:6/SM (d18:1/16:1) (d18:1/15:2-OH) 0.00031 −27.3 0.7377.8 62.2 PC 18:0/22:6/SM (d18:1/16:0) (d18:1/15:1-OH) 0.00161 −28.10.75 74.4 71.1 PC 18:0/22:6/SM (d18:1/15:0) (d18:1/14:1-OH) 0.00727−28.7 0.73 76.2 60.5 PC 18:0/22:6/SM (d18:1/18:0) 0.00565 −28.9 0.7269.8 64.9 PC 18:0/22:6/SM (d18:1/16:1) (d18:1/15:2-OH) 0.00018 −33.30.78 81.4 60.5 PC 18:0/22:6/SM (d18:1/18:1) 0.00074 −34.5 0.76 83.7 65.8

4c) Significant lipid-clinical concentration ratio markers for CVDpatients not undergoing statin treatment and not suffering fromdiabetes. Percentage Measurement name P-value change AUC SensitivitySpecificity Increased Cer(d18:1/16:0)/HDL cholesterol 0.00649 39.2 0.6970.9 63.0 GlcCer(d18:1/16:0)/apolipoprotein A-I 0.01107 32.2 CE 19:1oxCE 682.6/apolipoprotein A-I 32.0 0.66 63.6 66.7GlcCer(d18:1/18:0)/apolipoprotein A-I 0.00289 31.9 0.69 72.5 60.9GlcCer(d18:1/18:0)/HDL cholesterol 0.00816 31.2 0.68 70.6 60.9GlcCer(d18:1/16:0)/HDL cholesterol 0.01982 31.1GlcCer(d18:1/20:0)/apolipoprotein A-I 0.01983 21.5 0.66 68.6 60.9GlcCer(d18:1/18:0)/total cholesterol 0.00787 21.5 0.65 68.6 60.9Cer(d18:1/16:0)/apolipoprotein B 0.01020 20.3 0.66 61.8 60.9 DecreasedLacCer(d18:1/24:0)/supersensitive C-reactive protein −9.1 0.65 64.6 60.0CE 17:1/supersensitive C-reactive protein −12.3 0.64 61.5 61.9 SM(d18:1/16:0) (d18:1/15:1-OH)/supersensitive C- −13.0 0.64 60.0 61.4reactive protein GlcCer(d18:1/24:0)/supersensitive C-reactive protein−13.1 0.64 65.5 60.0 Gb3(d18:1/22:0)/supersensitive C-reactive protein−14.0 0.64 60.0 68.2 PC 18:0/20:3/apolipoprotein B 0.01774 −16.0 PC16:0/16:1/supersensitive C-reactive protein −16.2 0.64 63.0 63.4 PC16:0/22:6/total cholesterol 0.00719 −16.5 PC 16:0/22:6/apolipoprotein B0.00559 −19.0 PC 18:1/18:1/supersensitive C-reactive protein −19.0 0.6461.8 60.5 SM (d18:1/24:1) (d18:1/23:2-OH)/supersensitive C- −19.2 0.6665.5 65.9 reactive protein PC 18:0/18:2/supersensitive C-reactiveprotein −20.4 0.64 61.8 64.4 Cer(d18:1/24:1)/supersensitive C-reactiveprotein −20.5 0.64 60.0 62.2 Cer(d18:1/22:0)/supersensitive C-reactiveprotein −21.0 0.64 61.8 68.9 PC 18:0/22:6/triglycerides −21.4 0.67 60.564.1 SM (d18:1/18:0)/supersensitive C-reactive protein −22.3 0.64 63.660.5 PC 18:0/20:3/supersensitive C-reactive protein −22.4 0.65 66.7 60.0SM (d18:1124:0) (d18:1/23:1-OH)/supersensitive C- −22.8 0.65 63.0 61.4reactive protein PC 16:0/18:1/supersensitive C-reactive protein −22.90.64 61.8 62.2 SM (d18:1/23:1) (d18:1/22:2-OH)/total cholesterol 0.01384−23.0 PC 18:0/22:6/LDL cholesterol 0.00466 −23.1 0.73 76.7 61.5 PC18:0/22:6/total cholesterol 0.00079 −23.2 0.73 81.4 64.1 SM (d18:1/23:1)(d18:1/22:2-OH)/LDL cholesterol 0.01112 −23.8 SM (d18:1/23:1)(d18:1/22:2-OH)/apolipoprotein B 0.01067 −24.9 0.64 60.0 61.0 PC18:0/22:6/apolipoprotein B 0.00065 −25.2 0.74 81.4 64.1 SM (d18:1/23:0)(d18:1/22:1-OH)/supersensitive C- −27.0 0.65 63.0 61.4 reactive proteinPC 18:0/18:1/supersensitive C-reactive protein −27.2 0.64 63.0 60.0 LPC16:0/supersensitive C-reactive protein −27.7 0.65 63.6 62.2 PC16:0/22:6/supersensitive C-reactive protein −30.9 0.66 63.0 64.4 SM(d18:1/23:1) (d18:1/22:2-OH)/supersensitive C- −34.8 0.69 72.0 60.0reactive protein PC 18:0/22:6/lipoprotein(a) −46.6 0.66 63.9 71.4 PC16:0/18:0/supersensitive C-reactive protein 0.03865 −47.5 0.65 62.8 61.8

Table 5. Significant markers for CVD patients not undergoing statintreatment and having diabetes. Marker names, p-values, percentagechange, AUC, Specificity and Sensitivity are presented. Table 5a showssignificant lipid markers, Table 5b shows significant lipid-lipidconcentration ratio markers and Table 5c shows significantlipid-clinical concentration ratio markers.

5a) Significant lipid markers for CVD patients not undergoing statintreatment and having diabetes. Measurement Percentage Sen- name P-valuechange AUC sitivity Specificity Increased Gb3(d18:1/24:1) 0.02669 25.9Gb3(d18:1/16:0) 0.01318 17.6 Decreased PC O-16:0/20: −13.7 0.58 61.560.0 4-alkyl CE 20:4 0.02179 −14.0 0.61 64.2 60.0 CE 18:0 0.00754 −18.0

5b) Significant lipid-lipid concentration ratio markers for CVD patientsnot undergoing statin treatment and having diabetes. PercentageMeasurement name P-value change AUC Sensitivity Specificity IncreasedGb3(d18:1/24:1)/SM (d18:1/17:0) (d18:1/16:1-OH) 0.00674 44.5 0.69 69.260.5 Gb3(d18:1/24:1)/PC O-16:0/20:4-alkyl 0.00606 33.8 0.68 69.2 60.0Gb3(d18:1/16:0)/SM (d18:1/17:0) (d18:1/16:1-OH) 0.03447 32.6 0.66 63.562.8 Gb3(d18:1/24:1)/GlcCer(d18:1/24:0) 0.00369 31.5 0.67 75.0 62.0Gb3(d18:1/24:1)/GlcCer(d18:1/22:0) 0.00635 27.8 0.66 63.2 62.0Gb3(d18:1/22:0)/SM (d18:1/17:0) (d18:1/16:1-OH) 26.4 0.67 68.6 61.9Decreased CE 18:0/PC 18:0/18:2 −12.0 0.67 64.1 65.2 PC 18:0/18:2/PE18:0/18:2 −13.0 0.67 70.7 61.5 Cer(d18:1/24:0)/PE 18:0/18:2 −13.8 0.6565.5 61.5 CE 18:0/CE 18:1 0.00266 −15.6 CE 18:2/Gb3(d18:1/16:0) 0.00336−16.2 0.66 74.6 62.0 CE 16:0/Gb3(d18:1/16:0) 0.00265 −16.4 0.66 64.260.0 CE 18:0/PC 16:0/22:6 −17.5 0.68 70.3 60.0 CE 14:0/Gb3(d18:1/24:0)−18.9 0.66 67.7 61.2 CE 18:0/SM (d18:1/16:0) (d18:1/15:1-OH) 0.00313−19.1 CE 18:3/SM (d18:1/14:0) (d18:1/13:1-OH) 0.00287 −19.3 0.66 67.260.0 CE 18:0/PC 16:0/18:2 0.00185 −19.6 0.69 62.5 65.2Cer(d18:1/24:0)/PC O-16:0/18:1-alkyl 0.00226 −19.8 0.67 66.0 61.5 CE18:0/Gb3(d18:1/24:0) −20.6 0.66 61.7 60.0 CE 18:3/PC 16:0/18:1 0.00081−20.8 0.69 73.1 62.0 PC 18:0/20:3/PC O-16:0/18:1-alkyl 0.00849 −20.90.69 73.6 61.5 CE 14:0/PC 16:0/16:0 0.00266 −20.9 0.65 65.2 63.3 CE17:1/Gb3(d18:1/16:0) 0.00301 −21.1 0.66 68.3 60.9 CE 14:0/SM(d18:1/14:0) (d18:1/13:1-OH) 0.00090 −21.4 PC 18:0/20:3/PE 18:0/18:20.00794 −21.5 0.70 65.5 71.8 CE 18:0/SM (d18:1/14:0) (d18:1/13:1-OH)0.00120 −21.6 0.70 75.0 60.9 CE 14:0/PC 16:0/18:1 0.00110 −21.8 0.6668.2 60.0 CE 18:0/SM (d18:1/24:1) (d18:1/23:2-OH) −21.9 0.66 62.5 60.9CE 18:0/Cer(d18:1/16:0) 0.00203 −22.5 0.65 60.9 60.9 CE18:0/Cer(d18:1/24:1) 0.00347 −22.9 0.68 68.8 63.0 CE 18:0/PC 16:0/16:00.00085 −23.4 0.66 62.5 62.2 CE 18:0/PC 18:1/18:1 0.00151 −23.7 0.6763.5 61.4 CE 18:0/PC 16:0/18:1 0.00032 −24.1 0.69 67.2 60.9 CE 20:4/PCO-16:0/18:1-alkyl 0.00261 −24.1 CE 18:3/PE 18:0/18:2 0.00810 −24.2 0.6863.2 66.7 CE 20:4/Gb3(d18:1/16:0) 0.00070 −24.4 0.66 61.2 66.0 CE14:0/PE 18:0/18:2 0.00735 −24.9 0.68 75.0 61.5 CE 18:0/Cer(d18:1/26:1)0.00471 −25.0 0.66 62.3 68.4 CE 18:3/Gb3(d18:1/22:0) 0.00487 −25.4 0.6661.5 61.2 CE 14:0/Gb3(d18:1/16:0) 0.00189 −25.4 0.67 68.2 60.0 CE18:3/PC O-16:0/18:1-alkyl 0.00038 −25.7 0.70 75.0 66.7 CE18:3/Gb3(d18:1/16:0) 0.00180 −26.0 0.66 70.1 62.0 CE 14:0/PCO-16:0/18:1-alkyl 0.00041 −26.5 0.70 72.5 64.1 CE 20:4/Gb3(d18:1/18:0)0.00233 −26.6 0.66 68.3 61.7 CE 18:3/Gb3(d18:1/24:1) 0.00624 −27.3 0.6768.7 60.0 CE 14:0/Gb3(d18:1/24:1) 0.00652 −27.6 0.68 65.2 60.0 CE20:5/PC O-16:0/18:1-alkyl −28.0 0.67 68.6 64.1 CE 18:0/Gb3(d18:1/18:0)0.00246 −28.2 0.66 61.4 60.5 CE 18:0/Gb3(d18:1/16:0) 0.00056 −28.4 0.6864.1 63.0 CE 18:0/Gb3(d18:1/24:1) −34.6 0.69 67.2 60.9

5c) Significant lipid-clinical concentration ratio markers for CVDpatients not undergoing statin treatment and having diabetes. PercentageMeasurement name P-value change AUC Sensitivity Specificity Increased PC18:1/18:1/lipoprotein(a) 381.2 0.67 62.3 61.7 PCO-16:0/18:1-alkyl/lipoprotein(a) 221.1 0.68 70.2 63.2Gb3(d18:1/18:0)/lipoprotein(a) 0.01723 180.6 0.67 60.0 65.2 SM(d18:1/23:1) (d18:1/22:2-OH)/lipoprotein(a) 0.01616 179.1 0.67 63.8 60.0Gb3(d18:1/24:1)/lipoprotein(a) 0.01496 176.8Gb3(d18:1/16:0)/lipoprotein(a) 0.03006 174.6 0.67 64.5 61.2 PE18:0/18:2/lipoprotein(a) 0.01545 173.2 LacCer(d18:1/24:1)/lipoprotein(a)164.4 0.65 62.9 64.6 LacCer(d18:1/22:0)/lipoprotein(a) 158.5 0.64 62.961.2 Gb3(d18:1/22:0)/lipoprotein(a) 0.01598 148.3 CE 17:1/lipoprotein(a)0.04319 148.1 0.64 64.9 60.0 Gb3(d18:1/24:0)/lipoprotein(a) 0.04567131.6 0.67 63.8 62.5 PC 16:0/18:2/lipoprotein(a) 0.01114 128.9 PCO-18:0/18:2-alkyl/lipoprotein(a) 0.04267 125.5 0.65 61.8 60.0LacCer(d18:1/24:0)/lipoprotein(a) 117.2 0.64 60.3 60.5 PC17:0/18:2/lipoprotein(a) 116.6 0.65 66.1 60.5 SM(d18:1/18:0)/lipoprotein(a) 0.01151 108.7 CE 15:0/lipoprotein(a) 0.0325598.1 0.64 64.3 61.4 PC O-16:0/18:2-alkyl/lipoprotein(a) 70.4 0.65 67.361.0 Gb3(d18:1/24:1)/LDL cholesterol 0.00201 43.8Gb3(d18:1/24:1)/apolipoprotein B 0.00406 37.8 Gb3(d18:1/24:1)/totalcholesterol 0.00279 35.6 Gb3(d18:1/24:1)/apolipoprotein A-I 0.00640 35.3PC O-16:0/18:1-alkyl/LDL cholesterol 0.01994 34.1 0.66 60.4 64.1Gb3(d18:1/16:0)/LDL cholesterol 0.00024 33.2 0.69 64.7 60.0Gb3(d18:1/24:1)/HDL cholesterol 0.01160 32.0 Gb3(d18:1/22:0)/LDLcholesterol 0.00367 31.2 Gb3(d18:1/18:0)/LDL cholesterol 0.00054 30.3Gb3(d18:1/24:0)/LDL cholesterol 0.01420 29.8Gb3(d18:1/16:0)/apolipoprotein B 0.00125 27.4 0.67 67.6 60.0 PCO-16:0/18:1-alkyl/apolipoprotein B 0.00725 26.5 PCO-16:0/18:1-alkyl/triglycerides 26.3 0.61 62.3 61.5Gb3(d18:1/16:0)/total cholesterol 0.00060 25.5 0.68 66.2 62.0Gb3(d18:1/22:0)/apolipoprotein B 0.01357 25.3 PC 16:0/16:0/LDLcholesterol 0.01619 25.0 Gb3(d18:1/18:0)/apolipoprotein B 0.00545 24.6PC O-16:0/18:1-alkyl/total cholesterol 0.00448 24.1 SM (d18:1/24:1)(d18:1/23:2-OH)/LDL cholesterol 0.04799 24.0 0.62 62.7 62.0Gb3(d18:1/16:0)/triglycerides 23.8 0.60 64.7 60.0 PE 18:0/18:2/LDLcholesterol 23.4 0.62 65.5 61.5 Gb3(d18:1/22:0)/total cholesterol0.01269 22.9 PE 18:0/18:2/triglycerides 21.6 0.63 67.2 61.5Gb3(d18:1/18:0)/total cholesterol 0.01020 21.4 PE 18:0/18:2/totalcholesterol 0.01992 19.2 0.64 62.1 61.5 PC 16:0/18:2/LDL cholesterol17.9 0.60 61.8 64.0 PC 16:0/16:0/total cholesterol 0.01555 17.5 PE18:0/18:2/apolipoprotein B 15.0 0.64 65.5 61.5 SM (d18:1/14:0)(d18:1/13:1-OH)/total cholesterol 0.04182 11.0 0.62 61.2 60.0 DecreasedCE 14:0/HDL cholesterol −13.1 0.61 63.6 62.0 CE 14:0/supersensitiveC-reactive protein −36.8 0.62 60.6 60.0

TABLE 6 The preferred embodiment markers from CVD patients notundergoing statin treatment and not having diabetes. PercentageMeasurement name P-value change AUC Sensitivity Specificity LipidsIncreased CE 19:1 oxCE 682.6 27.7 0.65 70.5 66.7 GlcCer(d18:1/16:0)0.02176 22.7 SM (d18:1/18:1) 14.2 0.59 63.0 60.0 Decreased PC 18:0/22:60.00685 −20.3 0.67 74.4 61.5 SM (d18:1/23:1) (d18:1/22:2-OH) 0.01808−22.1 0.64 60.0 61.0 Lipid-lipid concentration ratios IncreasedCer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/22:2-OH) 0.00331 81.9 0.70 68.863.4 Cer(d18:1/16:0)/PC 18:0/22:6 0.00045 78.7 0.74 76.7 61.5Cer(d18:1/18:0)/PC 16:0/22:6 0.00259 74.5 0.71 67.3 67.4GlcCer(d18:1/16:0)/PC 18:0/22:6 0.00101 69.7 0.73 74.4 61.5GlcCer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/22:2-OH) 0.00462 68.5 0.7576.1 65.9 Cer(d18:1/16:0)/PC 16:0/22:6 0.00024 62.1 0.72 75.9 60.9Cer(d18:1/20:0)/SM (d18:1/23:1) (d18:1/22:2-OH) 0.00573 62.0 0.71 67.461.0 GlcCer(d18:1/18:0)/PC 16:0/22:6 0.00006 60.4 0.76 80.0 60.9 SM(d18:1/18:1)/SM (d18:1/23:1) (d18:1/22:2-OH) 60.2 0.76 81.6 63.4Cer(d18:1/20:0)/PC 16:0/22:6 0.00017 59.5 0.73 74.0 63.0 SM (d18:1/16:0)(d18:1/15:1-OH)/SM (d18:1/23:1) 57.1 0.73 74.0 63.4 (d18:1/22:2-OH) SM(d18:1/15:0) (d18:1/14:1-OH)/SM (d18:1/23:1) 55.1 0.73 78.0 63.4(d18:1/22:2-OH) GlcCer(d18:1/20:0)/SM (d18:1/23:1) (d18:1/22:2-OH) 54.90.71 78.3 61.0 Cer(d18:1/24:1)/PC 18:0/22:6 53.8 0.70 72.1 69.2 SM(d18:1/16:1) (d18:1/15:2-OH)/SM (d18:1/23:1) 52.9 0.75 82.0 61.0(d18:1/22:2-OH) GlcCer(d18:1/16:0)/PC 16:0/22:6 0.00081 52.6 0.72 75.960.9 LacCer(d18:1/22:0)/PC 16:0/22:6 0.00047 52.4 0.73 68.0 60.9 CE16:0/PC 18:0/22:6 0.00008 47.2 0.76 81.4 64.1 CE 18:2/PC 18:0/22:60.00025 43.5 0.75 67.4 64.1 Cer(d18:1/16:0)/Cer(d18:1/24:0) 42.0 0.7581.8 60.9 Decreased PC 18:0/22:6/SM (d18:1/16:1) (d18:1/15:2-OH) 0.00018−33.3 0.78 81.4 60.5 PC 18:0/22:6/SM (d18:1/18:1) 0.00074 −34.5 0.7683.7 65.8 Lipid-clinical concentration ratios IncreasedCer(d18:1/16:0)/HDL cholesterol 0.00649 39.2 0.69 70.9 63.0 Decreased PC18:0/22:6/LDL cholesterol 0.00466 −23.1 0.73 76.7 61.5 PC18:0/22:6/total cholesterol 0.00079 −23.2 0.73 81.4 64.1 PC18:0/22:6/apolipoprotein B 0.00065 −25.2 0.74 81.4 64.1

TABLE 7 The preferred embodiment markers from CVD patients notundergoing statin treatment and having diabetes. Percentage Measurementname P-value change AUC Sensitivity Specificity Lipids IncreasedGb3(d18:1/24:1) 0.02669 25.9 Gb3(d18:1/16:0) 0.01318 17.6 Decreased PCO-16:0/20:4-alkyl −13.7 0.58 61.5 60.0 CE 20:4 0.02179 −14.0 0.61 64.260.0 CE 18:0 0.00754 −18.0 Lipid-lipid concentration ratios IncreasedGb3(d18:1/24:1)/SM (d18:1/17:0) (d18:1/16:1-OH) 0.00674 44.5 0.69 69.260.5 Gb3(d18:1/16:0)/SM (d18:1/17:0) (d18:1/16:1-OH) 0.03447 32.6 0.6663.5 62.8 Gb3(d18:1/24:1)/GlcCer(d18:1/24:0) 0.00369 31.5 0.67 75.0 62.0Decreased CE 18:3/PC 16:0/18:1 0.00081 −20.8 0.69 73.1 62.0 CE 18:0/SM(d18:1/14:0) (d18:1/13:1-OH) 0.00120 −21.6 0.70 75.0 60.9 CE 14:0/PE18:0/18:2 0.00735 −24.9 0.68 75.0 61.5 CE 18:3/PC O-16:0/18:1-alkyl0.00038 −25.7 0.70 75.0 66.7 CE 18:0/Gb3(d18:1/16:0) 0.00056 −28.4 0.6864.1 63.0 CE 18:0/Gb3(d18:1/24:1) −34.6 0.69 67.2 60.9 Lipid-clinicalconcentration ratios Increased PC 18:1/18:1/lipoprotein(a) 381.2 0.6762.3 61.7 Gb3(d18:1/18:0)/lipoprotein(a) 0.01723 180.6 0.67 60.0 65.2Gb3(d18:1/16:0)/lipoprotein(a) 0.03006 174.6 0.67 64.5 61.2LacCer(d18:1/24:1)/lipoprotein(a) 164.4 0.65 62.9 64.6 Decreased CE14:0/supersensitive C-reactive protein −36.8 0.62 60.6 60.0

The preferred lipid molecules of the invention were selected as follows:a) it was likely to be biologically meaningful, b) it preferably belongsto a family of lipids that are behaving similarly, c) it is expressed inmeaningful & measurable concentrations, d) it has very significantp-value or good AUC-value (>0.65) and for most also the %-change issubstantial (>20%), and e) it appeared significant in different tests

TABLE 8 Lipid markers generated with logistic modeling. CVD high riskmarkers for CVD patients not CVD high risk markers for CVD patients noton statin treatment and not having diabetes on statin treatment andhaving diabetes Lipid Direction of change Lipid Direction of changeGlcCer 18:1/16:0 increased Gb3 18:1/16:0 increased CE 20:4 increasedGlcCer 18:1/16:0 increased LacCer(d18:1/16:0) increased LacCer 18:1/16:0increased Cer 18:1/16:0 increased PC 16:0/22:6 increased CE 16:0increased CE 14:0 decreased SM 18:1/16:0 increased CE 22:6 decreased LPC16:0 decreased CE 18:3 decreased PC 16:0/22:6 decreased GlcCer 18:1/18:0decreased SM 18:1/24:0 decreased SM 18:1/24:0 decreased Lipidomicanalysis proved to be efficient in identifying novel plasma biomarkersfor CVD complications.

Molecular lipid to molecular lipid ratio could be an important indicatorof cellular lipid metabolism including e.g., enzyme activities in thelipid metabolism pathways. Thus, these ratios may provide moreinformation as the absolute plasma concentrations of the molecularlipids alone. As the absolute molecular lipid plasma concentrationdifferences in general between healthy individuals and atheroscleroticpatients seem to be between 30-70%, it might be reasonable to calculateand use different ratios instead of absolute concentrations only. Aslipoprotein particles (e.g. LDL, HDL and VLDL) are serving as carriersfor most of the lipids in the blood stream it is appropriate to relatemolecular lipid concentrations to lipoprotein data. Thus, the molecularlipid to HDL-cholesterol, LDL-cholesterol, apolipoprotein A-I andapolipoprotein B ratios were calculated. In fact, a number of ratiosbetween the concentrations of different molecular lipids outperformedabsolute plasma concentrations as disease biomarkers in CVD patients.

As the detected lipids are carried in the lipoprotein particles (LDL,VLDL and HDL) it is obvious that the corresponding lipoprotein fractionconcentrations will even improve the prediction potential of molecularlipids from the results of the present study in total serum/plasmasamples.

The lipid lowering drug efficiency measurements have so far been basedon LDL-C and HDL-C assays. As the inventors have herein observed morepotential biomarkers that predict the development of high-risk CVDcomplications better than these classical analyses, future drugefficiency profiling should be based on new sensitive and specificbiomarkers that are more directly related to the risk of severeCVD-related complications rather than to LDL-C.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific embodiments described herein both in the Examples in the bodyof the entire patent description. Such equivalents are considered to bewithin the scope of this invention and are covered by the followingclaims.

The invention claimed is:
 1. A method for determining whether a subjectnot undergoing statin treatment who is not suffering from type 2diabetes mellitus is at risk to develop one or more CardiovascularDisease (CVD) complications, comprising: a) determining in a sample fromsaid subject one or more lipid-lipid concentration ratio(s), wherein(an) increased lipid-lipid concentration ratio in said sample, whencompared to a control sample is (are) indicative of said subject havingan increased risk of developing one or more CVD complications, whereinthe one or more lipid-lipid concentration ratio(s) whose increase(s) is(are) compared to the control is (are) selected from:Cer(d18:1/16:0)/Cer(d18:1/22:0) and Cer(d18:1/16:0)/Cer(d18:1/24:0); orb) determining in a sample from said subject one or more lipid-clinicalconcentration ratio(s), wherein (an) increased or decreasedlipid-clinical concentration ratio(s) in said sample, when compared to acontrol sample is (are) indicative of said subject having an increasedrisk of developing one or more CVD complications, wherein the one ormore lipid-clinical concentration ratio(s) whose increase(s) is (are)compared to the control is selected from: Cer(d18:1/16:0)/HDLcholesterol and Cer(d18:1/16:0)/apoliprotein B; and wherein the one ormore lipid-clinical concentration ratio(s) whose decrease(s) is (are)compared to the control is (are) selected from:Cer(d18:1/24:1)/supersensitive C-reactive protein andCer(d18:1/22:0)/supersensitive C-reactive protein, and wherein saiddetermining comprises determining at least two lipid-lipid concentrationratios or at least two lipid-clinical concentration ratios or acombination of at least one lipid-lipid concentration ratio and at leastone lipid-clinical concentration ratio.
 2. The method of claim 1,wherein determining the lipid-lipid concentration ratio(s) or thelipid-clinical concentration ratio(s) is done using mass spectrometry.3. The method of claim 1, wherein a) said CVD is characterized bycoronary artery disease, peripheral artery disease, a stroke and/or CVDdeath; and/or b) said CVD is atherosclerosis-induced; and/or c) saidsubject has atherosclerosis; or d) said subject does not haveatherosclerosis.
 4. The method of claim 1, wherein a) the method furthercomprises determining the serum or plasma level of total cholesterol,low-density lipoprotein cholesterol (LDL-C), high-density lipoproteincholesterol (HDL-C), Apolipoprotein B (ApoB) and/or Apolipoprotein C-III(ApoC-III) in said sample; and/or b) the subject does not have elevatedserum or plasma levels of one or more of total cholesterol, low-densitylipoprotein cholesterol (LDL-C), Apolipoprotein C-III (ApoC-III) orApolipoprotein B (ApoB), or a decreased serum level of HDL-cholesterol(HDL-C).
 5. The method of claim 1, wherein a) the sample is blood,plasma, serum, urine or tissue, or a lipoprotein fraction thereof;and/or b the lipid-lipid concentration ratio(s) or the lipid-clinicalconcentration ratio(s) is (are) determined by using mass spectrometry,nuclear magnetic resonance spectroscopy, fluorescence spectroscopy ordual polarisation interferometry, a high performance separation methodan immunoassay and/or with a binding moiety capable of specificallybinding the analyte.
 6. The method of claim 1, wherein the one or moreCVD complications are selected from CVD death and acute myocardialinfarction (AMI).
 7. The method of claim 1, wherein the subject is atrisk to develop or has suffered from one or more CVD complications. 8.The method of claim 1, wherein the control sample is from (a) CoronaryArtery Disease (CAD) patient(s) or a group of CAD patients, wherein theCAD patient(s) or group of CAD patients has/have no history of major CVDevents and is/are not undergoing statin treatment; wherein the controlsample is blood, plasma, serum, urine or tissue, or a lipoproteinfraction thereof.
 9. The method of claim 1, wherein the sample is blood,serum, or plasma and the one or more lipid-lipid concentration ratio(s)or the one or more lipid-clinical concentration ratio(s) is (are)determined by using mass spectrometry.
 10. The method of claim 9,further comprising a step of obtaining the sample from the subject. 11.The method of claim 9, further comprising a step of extracting lipidsfrom the blood, serum, or plasma sample.
 12. The method of claim 9,wherein the one or more CVD complications are selected from CVD deathand acute myocardial infarction (AMI).
 13. The method of claim 10,wherein the one or more CVD complications are selected from CVD deathand acute myocardial infarction (AMI).
 14. The method of claim 11,wherein the one or more CVD complications are selected from CVD deathand acute myocardial infarction (AMI).
 15. The method of claim 1,wherein the method further comprises: (d) treating the subject, if thedetermining steps indicate the subject has an increased risk ofdeveloping one or more CVD complications.
 16. A method of treating orpreventing one or more CVD complications in a subject who is notundergoing statin treatment and who is not suffering from type 2diabetes mellitus, the method comprising: administering a treatment tothe subject, wherein, prior to administering the treatment, the subjecthas been identified as being at risk to develop one or more CVDcomplications by the method according to claim
 1. 17. A method oftreating or preventing one or more CVD complications in a subject who isnot undergoing statin treatment and who is not suffering from type 2diabetes mellitus, the method comprising: determining whether thesubject is at risk to develop one or more CVD complications according tothe method of claim 1; treating the subject, if the subject has beenidentified as being at risk for CVD complications.
 18. The methodaccording to claim 1 further comprising: determining in a sample fromsaid subject the concentration of a lipid, wherein an increased lipidconcentration in said sample, when compared to a control sample isindicative of said subject having an increased risk of developing one ormore CVD complications, wherein the lipid whose increase inconcentration is compared to the control is Cer(d18:1/16:0).